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1.
Folia Histochem Cytobiol ; 57(4): 179-187, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31840794

RESUMO

INTRODUCTION: Stomach hyperacidity leads to damage of the mucus/bicarbonate barrier, ulcerations and the development of stomach cancer. Key regulators of the mucosal barrier/luminal acid balance are neurotransmitters secreted by intramural neurons. The aim of the current study was to determine the expression of gastric neuropeptides and nNOS in the porcine stomach following hydrochloric acid instillation. We report on increased expression of enteric neurotransmitters involved in adaptive reaction to an experimentally-induced hyperacidity state. MATERIAL AND METHODS: The investigation was conducted on eight 12-18 kg pigs. The influence of intragastric infusion of hydrochloric acid on the expression of cocaine- and amphetamine-regulated transcript peptide (CART), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), substance P (SP) and galanin (GAL) in the submucous and myenteric gastric neurons of the pig has been studied with double immunofluorescence. RESULTS: A mimicked hyperacidity state significantly increased the proportion of enteric neurons immunoreactive to CART, nNOS, VIP, PACAP, SP and GAL in the submucous gastric neurons. In the myenteric plexus, a significant increase of the number of VIP-, CART- and GAL-immunoreactive (IR) neurons was found. Similarly, the percentage of myenteric nNOS-IR and PACAP-IR neurons tended to increase, while the fraction of SP-IR cells did not change. CONCLUSIONS: Stomach hyperacidity modifies the expression of the studied neurotransmitters in a specific way depending on the location of the neurons in particular plexuses of the stomach. Increased numbers of neurons expressing CART, nNOS, VIP, PACAP, SP and GAL clearly indicate their regulatory engagement in the restoration of the physiological gastric balance following hyperacidity.


Assuntos
Ácido Clorídrico/farmacologia , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Estômago/inervação , Plexo Submucoso/metabolismo , Animais , Feminino , Ácido Clorídrico/administração & dosagem , Infusões Parenterais , Plexo Mientérico/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/metabolismo , Estômago/efeitos dos fármacos , Plexo Submucoso/efeitos dos fármacos , Suínos
2.
Int J Mol Sci ; 21(1)2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31861419

RESUMO

The digestive tract, especially the small intestine, is one of the main routes of acrylamide absorption and is therefore highly exposed to the toxic effect of acrylamide contained in food. The aim of this experiment was to elucidate the effect of low (tolerable daily intake-TDI) and high (ten times higher than TDI) doses of acrylamide on the neurochemical phenotype of duodenal enteric nervous system (ENS) neurons using the pig as an animal model. The experiment was performed on 15 immature gilts of the Danish Landrace assigned to three experimental groups: control (C) group-pigs administered empty gelatine capsules, low dose (LD) group-pigs administered capsules with acrylamide at the TDI dose (0.5 µg/kg body weight (b.w.)/day), and the high dose (HD) group-pigs administered capsules with acrylamide at a ten times higher dose than the TDI (5 µg/kg b.w./day) with a morning feeding for 4 weeks. Administration of acrylamide, even in a low (TDI) dose, led to an increase in the percentage of enteric neurons immunoreactive to substance P (SP), calcitonin gene-related peptide (CGRP), galanin (GAL), neuronal nitric oxide synthase (nNOS), and vesicular acetylcholine transporter (VACHT) in the porcine duodenum. The severity of the changes clearly depended on the dose of acrylamide and the examined plexus. The obtained results suggest the participation of these neuroactive substances in acrylamide-inducted plasticity and the protection of ENS neurons, which may be an important line of defence from the harmful action of acrylamide.


Assuntos
Acrilamida/farmacologia , Duodeno/inervação , Duodeno/metabolismo , Sistema Nervoso Entérico/efeitos dos fármacos , Sistema Nervoso Entérico/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Animais , Imunofluorescência , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/metabolismo , Plexo Submucoso/efeitos dos fármacos , Plexo Submucoso/metabolismo , Suínos
3.
World J Gastroenterol ; 23(41): 7359-7368, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-29151690

RESUMO

AIM: To investigate the intestinal segment-specific effects of diabetes and insulin replacement on the density of different subpopulations of submucous neurons. METHODS: Ten weeks after the onset of type 1 diabetes samples were taken from the duodenum, ileum and colon of streptozotocin-induce diabetic, insulin-treated diabetic and sex- and age-matched control rats. Whole-mount preparations of submucous plexus were prepared from the different gut segments for quantitative fluorescent immunohistochemistry. The following double-immunostainings were performed: neuronal nitric oxide synthase (nNOS) and HuC/D, heme oxygenase (HO) 1 and peripherin, as well as HO2 and peripherin. The density of nNOS-, HO1- and HO2-immunoreactive (IR) neurons was determined as a percentage of the total number of submucous neurons. RESULTS: The total number of submucous neurons and the proportion of nNOS-, HO1- and HO2-IR subpopulations were not affected in the duodenal ganglia of control, diabetic and insulin-treated rats. While the total neuronal number did not change in either the ileum or the colon, the density of nitrergic neurons exhibited a 2- and 3-fold increase in the diabetic ileum and colon, respectively, which was further enhanced after insulin replacement. The presence of HO1- and HO2-IR submucous neurons was robust in the colon of controls (38.4%-50.8%), whereas it was significantly lower in the small intestinal segments (0.0%-4.2%, P < 0.0001). Under pathophysiological conditions the only alteration detected was an increase in the ileum and a decrease in the colon of the proportion of HO-IR neurons in insulin-treated diabetic animals. CONCLUSION: Diabetes and immediate insulin replacement induce the most pronounced region-specific alterations of nNOS-, HO1- and HO2-IR submucous neuronal density in the distal parts of the gut.


Assuntos
Colo/inervação , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/fisiopatologia , Duodeno/inervação , Íleo/inervação , Neurônios/fisiologia , Animais , Contagem de Células , Colo/efeitos dos fármacos , Colo/patologia , Colo/fisiopatologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/patologia , Duodeno/patologia , Duodeno/fisiopatologia , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Íleo/efeitos dos fármacos , Íleo/patologia , Íleo/fisiopatologia , Imuno-Histoquímica , Insulina/farmacologia , Insulina/uso terapêutico , Masculino , Plexo Mientérico/citologia , Plexo Mientérico/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/metabolismo , Ratos , Ratos Wistar , Estreptozocina/toxicidade , Plexo Submucoso/citologia , Plexo Submucoso/efeitos dos fármacos
4.
J Histochem Cytochem ; 64(9): 530-45, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27389702

RESUMO

Oxaliplatin, currently used for treatment of colorectal and other cancers, causes severe gastrointestinal side effects, including nausea, vomiting, diarrhea, and constipation that are attributed to mucosal damage. However, delayed onset and long-term persistence of these side effects suggest that damage to the enteric nervous system (ENS) regulating physiological function of the gastrointestinal tract may also occur. The ENS comprises myenteric and submucosal neurons and enteric glial cells (EGCs). This study aimed to investigate the effects of oxaliplatin treatment on enteric neurons and EGCs within the mouse ileum. BALB/c mice received repeated intraperitoneal injections of oxaliplatin (3 mg/kg, 3 injections/week). Tissues were collected 3, 7, 14, and 21 days from the commencement of treatment. Decreases in glial fibrillary acidic protein-immunoreactive (IR) EGCs and protein gene product 9.5/ß-Tubulin III-IR neurons as well as increase in s100ß-IR EGCs after chronic oxaliplatin administration were observed in both the myenteric and submucosal plexi. Changes in EGCs were further observed in cross-sections of the ileum at day 14 and confirmed by Western blotting. Alterations in EGCs correlated with loss of myenteric and submucosal neurons in the ileum from oxaliplatin-treated mice. These changes to the ENS may contribute to the mechanisms underlying gastrointestinal side effects associated with oxaliplatin treatment.


Assuntos
Antineoplásicos/efeitos adversos , Íleo/efeitos dos fármacos , Plexo Mientérico/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Compostos Organoplatínicos/efeitos adversos , Piridinas/efeitos adversos , Plexo Submucoso/efeitos dos fármacos , Animais , Proteína Glial Fibrilar Ácida/metabolismo , Íleo/inervação , Íleo/patologia , Masculino , Camundongos Endogâmicos BALB C , Plexo Mientérico/patologia , Neuroglia/patologia , Neurônios/patologia , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Plexo Submucoso/patologia
5.
Neurogastroenterol Motil ; 28(9): 1438-42, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27098706

RESUMO

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.


Assuntos
Sistema Nervoso Entérico/efeitos dos fármacos , Frutose/administração & dosagem , Neurônios Serotoninérgicos/efeitos dos fármacos , Serotonina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Plexo Submucoso/efeitos dos fármacos , Animais , Cálcio/metabolismo , Dieta , Sistema Nervoso Entérico/metabolismo , Camundongos , Neurônios Serotoninérgicos/metabolismo , Plexo Submucoso/metabolismo
6.
PLoS One ; 10(7): e0133350, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26230272

RESUMO

In the porcine colon, the submucous plexus is divided into an inner submucous plexus (ISP) on the epithelial side and an outer submucous plexus (OSP) on the circular muscle side. Although both plexuses are probably involved in the regulation of epithelial functions, they might differ in function and neurochemical coding according to their localization. Therefore, we examined expression and co-localization of different neurotransmitters and neuronal markers in both plexuses as well as in neuronal fibres. Immunohistochemical staining was performed on wholemount preparations of ISP and OSP and on cryostat sections. Antibodies against choline acetyltransferase (ChAT), substance P (SP), somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), neuronal nitric oxide synthase (nNOS) and the pan-neuronal markers Hu C/D and neuron specific enolase (NSE) were used. The ISP contained 1,380 ± 131 ganglia per cm2 and 122 ± 12 neurons per ganglion. In contrast, the OSP showed a wider meshwork (215 ± 33 ganglia per cm2) and smaller ganglia (57 ± 3 neurons per ganglion). In the ISP, 42% of all neurons expressed ChAT. About 66% of ChAT-positive neurons co-localized SP. A small number of ISP neurons expressed SOM. Chemical coding in the OSP was more complex. Besides the ChAT/±SP subpopulation (32% of all neurons), a nNOS-immunoreactive population (31%) was detected. Most nitrergic neurons were only immunoreactive for nNOS; 10% co-localized with VIP. A small subpopulation of OSP neurons was immunoreactive for ChAT/nNOS/±VIP. All types of neurotransmitters found in the ISP or OSP were also detected in neuronal fibres within the mucosa. We suppose that the cholinergic population in the ISP is involved in the control of epithelial functions. Regarding neurochemical coding, the OSP shares some similarities with the myenteric plexus. Because of its location and neurochemical characteristics, the OSP may be involved in controlling both the mucosa and circular muscle.


Assuntos
Colo/inervação , Plexo Submucoso/anatomia & histologia , Plexo Submucoso/metabolismo , Sus scrofa/anatomia & histologia , Sus scrofa/metabolismo , Animais , Colina O-Acetiltransferase/metabolismo , Colchicina/farmacologia , Colo/anatomia & histologia , Colo/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Neurônios/classificação , Neurônios/metabolismo , Neuropeptídeo Y/metabolismo , Neurotransmissores/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Somatostatina/metabolismo , Especificidade da Espécie , Plexo Submucoso/efeitos dos fármacos , Substância P/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo
8.
Neuropharmacology ; 95: 83-99, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25724083

RESUMO

RATIONALE: The role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. EXPERIMENTAL APPROACH: LSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs). RESULTS: Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-ß,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPßS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,ß-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 >> P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R. CONCLUSIONS: Purines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology.


Assuntos
Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Receptores Purinérgicos/metabolismo , Plexo Submucoso/efeitos dos fármacos , Plexo Submucoso/fisiologia , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Colectomia , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/fisiologia , Humanos , Imuno-Histoquímica , Purinérgicos/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Imagens com Corantes Sensíveis à Voltagem
9.
Neurogastroenterol Motil ; 25(8): 677-85, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23593931

RESUMO

BACKGROUND: 5-HT3 antagonists, such as ondansetron (Zofran), retard colonic transit and provide effective relief of symptoms of chronic diarrhea and diarrhea-predominant irritable bowel syndrome (IBS), but the mechanism by which ondansetron retards transit is unclear. What is clear is that the frequency of colonic migrating motor complexes (CMMCs) is reduced by ondansetron, which could account for reduced transit. Our aim was to determine whether an acute depletion of 5-HT from enteric neurons would inhibit spontaneous CMMCs; and determine whether the sensitivity of ondansetron to reduce CMMC frequency would change in a 5-HT-depleted preparation. METHODS: Mice were injected with reserpine, 24 h prior to euthanasia to deplete neuronally synthesized 5-HT. Mechanical recordings were made from proximal and mid-distal regions of isolated whole mouse colon. Immunohistochemical staining for 5-HT was used to detect neuronal 5-HT. KEY RESULTS: Reserpine depleted all detectable 5-HT from enteric nerves. In whole colons, with mucosa and submucosal plexus removed, the frequency and amplitude of spontaneous CMMCs was not different between groups treated with or without reserpine. Surprisingly, in mucosa and submucosal plexus-free preparations, ondansetron was equally or significantly more effective at inhibiting CMMC frequency compared with control preparations (containing 5-HT). Reserpine pretreatment had no effect on the sensitivity of ondansetron to inhibit CMMCs. CONCLUSIONS & INFERENCES: Endogenous 5-HT in enteric neurons (or the mucosa) is not required for the spontaneous generation or propagation of CMMCs. Furthermore, the primary mechanism by which ondansetron inhibits CMMC frequency is not mediated via the mucosa, submucosal plexus or 5-HT in myenteric neurons.


Assuntos
Colo/efeitos dos fármacos , Complexo Mioelétrico Migratório/efeitos dos fármacos , Ondansetron/farmacologia , Antagonistas da Serotonina/farmacologia , Serotonina , Plexo Submucoso/efeitos dos fármacos , Animais , Colo/metabolismo , Cobaias , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/metabolismo , Complexo Mioelétrico Migratório/fisiologia , Serotonina/deficiência , Plexo Submucoso/metabolismo
10.
Neurogastroenterol Motil ; 25(5): 439-47, e302, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23490018

RESUMO

BACKGROUND: Beneficial effects of ginger in the treatment of gastrointestinal (GI) problems and chemotherapy-induced nausea and vomiting are well accepted. In rodents, the action of ginger seems to be mediated by the inhibition of 5-HT3 receptors, which are established targets to combat emesis and irritable bowel syndrome. METHODS: Heterologously expressed human 5-HT3 A or 5-HT3 AB receptors were characterized by means of Ca(2+) influx studies using HEK293 cells. Complementing Ca(2+) measurements in Fluo-4-AM-stained whole-mount preparations of the human submucous plexus were carried out. Furthermore, [3H]GR65630 binding assays were performed to reveal the mode of action of ginger and its pungent compounds. KEY RESULTS: We show for the first time that ginger extracts and its pungent arylalkane constituents concentration-dependently inhibit activation of human 5-HT3 receptors. Ginger extracts inhibited both receptors with increasing content of pungent compounds, confirming that these are part of ginger's active principle. Inhibition potencies of the arylalkanes 6-gingerol and 6-shogaol on both receptors were in the low micromolar range. A lipophilic ginger extract and 6-gingerol had no influence on 5-HT potency, but reduced the 5-HT maximum effect, indicating non-competitive inhibition. The non-competitive action was confirmed by [(3) H]GR65630 binding, showing that the ginger extract did not displace the radioligand from 5-HT3 A and 5-HT3 AB receptors. The potential relevance of the inhibitory action of ginger on native 5-HT3 receptors in the gut was confirmed in whole-mount preparations of the human submucous plexus. While a general neurotoxic effect of 6-gingerol was ruled out, it inhibited the 2-methyl-5-HT-mediated activation of 5-HT3 receptors residing on enteric neurons. CONCLUSIONS & INFERENCES: Our findings may encourage the use of ginger extracts to alleviate nausea in cancer patients receiving chemotherapy and to treat functional GI disorders.


Assuntos
Catecóis/farmacologia , Álcoois Graxos/farmacologia , Neurônios/efeitos dos fármacos , Extratos Vegetais/farmacologia , Antagonistas do Receptor 5-HT3 de Serotonina/farmacologia , Plexo Submucoso/efeitos dos fármacos , Gengibre/química , Células HEK293 , Humanos , Neurônios/metabolismo , Receptores 5-HT3 de Serotonina/metabolismo , Proteínas Recombinantes/metabolismo , Plexo Submucoso/metabolismo
11.
Brain Behav Immun ; 30: 115-24, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23369733

RESUMO

BACKGROUND: Irritable bowel syndrome (IBS) is a common disorder of the gut with symptoms such as diarrhoea, constipation, abdominal pain and bloating, that are frequently exacerbated by stress. Circulating levels of the pro-inflammatory cytokine, interleukin-6 (IL-6), which can activate colonic enteric neurons, are elevated in IBS patients. These studies aim to explore the relationship between IL-6 and the stress peptide, corticotropin-releasing factor (CRF) in colonic submucosal neurons. METHODS: Calcium imaging, Ussing chamber electrophysiology and immunohistochemistry were conducted on rat distal colons to investigate potential crosstalk between IL-6 and CRF. KEY RESULTS: Colonic secretions from the maternal separation rat model of IBS stimulated increases in intracellular calcium in naïve submucosal neurons via CRF1 receptors (n=15, p<0.05). Moreover, IL-6 (n=50, p<0.01) but not IL-1ß (n=46, p>0.05) or TNFα (n=46, p>0.05) potentiated the CRF-evoked calcium response. CRF (1µM, 1h, n=5) stimulation also induced colonic secretion of IL-6 and inhibited the pro-secretory effects of IL-6 on colonic ion transfer (n=12). CONCLUSIONS AND INFERENCES: These studies demonstrate the modulatory effects of CRF on colonic IL-6 secretion, neuronal activation and secretory function. These findings may provide an insight into the molecular mechanisms underlying symptom flares in IBS during periods of high stress.


Assuntos
Colo/metabolismo , Hormônio Liberador da Corticotropina/farmacologia , Interleucina-6/farmacologia , Síndrome do Intestino Irritável/metabolismo , Neurônios/metabolismo , Plexo Submucoso/metabolismo , Animais , Cálcio/metabolismo , Colo/efeitos dos fármacos , Colo/fisiopatologia , Hormônio Liberador da Corticotropina/metabolismo , Feminino , Interleucina-6/metabolismo , Síndrome do Intestino Irritável/fisiopatologia , Masculino , Privação Materna , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Interleucina-6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Plexo Submucoso/efeitos dos fármacos , Plexo Submucoso/fisiopatologia
12.
Pharmazie ; 68(11): 877-81, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24380236

RESUMO

Previously, we found that the naturally occurring stilbene compound resveratrol (RES) could potentiate cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity. Because some wild-type CFTR activators also potentiate its mutant forms, we investigated effect of RES on the two most common forms of CF-related mutation (deltaF508 and G551D-CFTR). Cell-based fluorescence studies indicated that RES dose-dependently potentiated both deltaF508 and G551D mutant CFTR Cl- channel activities. Transepithelial Cl- currents were stimulated by RES in deltaF508 and G551D mutant CFTR-expressing FRT cells. Further excised inside-out patch-clamp measurements revealed that RES significantly induced the chloride current of deltaF508 and G551D mutant CFTRs by increasing the open time of the channels. In ex vivo studies, RES stimulated fluid secretion in mouse trachea by optical measurement of single gland secretion. These data suggested that RES is a potent deltaF508 and G551D mutant CFTR potentiator, and RES may present a novel class of therapeutic lead compounds in treating cystic fibrosis.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Estilbenos/farmacologia , Animais , Linhagem Celular , Iodetos/química , Mutação/genética , Mutação/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Endogâmicos F344 , Resveratrol , Plexo Submucoso/efeitos dos fármacos , Plexo Submucoso/metabolismo
13.
Neurogastroenterol Motil ; 24(12): 1134-e572, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22963673

RESUMO

BACKGROUND: We previously showed that colonic mucosal biopsy supernatants from patients with irritable bowel syndrome (IBS) activate neurons of the human submucous plexus, an area with densely packed immune cells. Based on the concept that mucosa-nerve signaling is altered in IBS, we tested in this study whether the nerve sensitizing effect of IBS mucosal biopsy supernatants is more prominent in the submucous than myenteric plexus. METHODS: Fast neuroimaging with the voltage-sensitive dye Di-8-ANEPPS was used to record activity of guinea-pig submucous and myenteric neurons after application of constipation (C)- and diarrhea (D)-IBS supernatants (three each) and four supernatants from healthy control subjects. Results are based on recordings from 4731 neurons. KEY RESULTS: Control supernatants did not evoke significant responses in submucous or myenteric neurons. In contrast, all IBS supernatants evoked a significant spike discharge (median 3.6 Hz) in 46% of submucous neurons. This activation was significantly stronger than in the myenteric plexus where even twice the amount of supernatants evoked a lower spike frequency (median 2.1Hz) in only 8.5% of neurons. Pharmacological studies revealed serotonin, histamine, and proteases as components mediating neuronal activation. Individual application of these components revealed that only serotonin evoked a significantly stronger activation of submucous compared with myenteric neurons. CONCLUSIONS & INFERENCES: Direct neuronal activation by IBS mucosal biopsy supernatants is primarily a feature of submucous rather than myenteric neurons. This is associated with a stronger excitation of submucous neurons by serotonin. The plexus-specific effects support the concept that altered mucosa-nerve signaling underlies disturbances in IBS.


Assuntos
Meios de Cultivo Condicionados/farmacologia , Mucosa Intestinal/metabolismo , Síndrome do Intestino Irritável/metabolismo , Neurônios/efeitos dos fármacos , Plexo Submucoso/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Adulto , Animais , Biópsia , Eletrofisiologia , Feminino , Cobaias , Humanos , Masculino , Pessoa de Meia-Idade , Plexo Mientérico/efeitos dos fármacos , Adulto Jovem
14.
Am J Physiol Endocrinol Metab ; 303(9): E1142-50, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22932783

RESUMO

The hormone relaxin exerts a variety of functions on the smooth muscle of reproductive and nonreproductive organs, most of which occur through a nitric oxide (NO)-mediated mechanism. In the stomach and ileum, relaxin causes muscle relaxation by modulating the activity and expression of different nitric oxide synthase (NOS) isoforms region-dependently. Nothing is known on the effects of relaxin in the colon, the gut region expressing the highest number of neuronal (n) NOSß-immunoreactive neurons and mainly involved in motor symptoms of pregnancy and menstrual cycle. Therefore, we studied the effects of relaxin exposure in the mouse proximal colon in vitro evaluating muscle mechanical activity and NOS isoform expression. The functional experiments showed that relaxin decreases muscle tone and increases amplitude of spontaneous contractions; the immunohistochemical results showed that relaxin increases nNOSß and endothelial (e) NOS expression in the neurons and decreases nNOSα and eNOS expression in the smooth muscle cells (SMC). We hypothesized that, in the colon, relaxin primarily increases the activity and expression of nNOSß and eNOS in the neurons, causing a reduction of the muscle tone. The downregulation of nNOSα and eNOS expression in the SMC associated with increased muscle contractility could be the consequence of continuous exposue of these cells to the NO of neuronal origin. These findings may help to better understand the physiology of NO in the gastrointestinal tract and the role that the "relaxin-NO" system plays in motor disorders such as functional bowel disease.


Assuntos
Colo/metabolismo , Contração Muscular , Músculo Liso/metabolismo , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Relaxina/metabolismo , Anestésicos Locais/farmacologia , Animais , Colo/irrigação sanguínea , Colo/citologia , Colo/inervação , Colo Ascendente/citologia , Colo Ascendente/efeitos dos fármacos , Colo Ascendente/inervação , Colo Ascendente/metabolismo , Colo Transverso/citologia , Colo Transverso/efeitos dos fármacos , Colo Transverso/inervação , Colo Transverso/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Guanilato Ciclase/antagonistas & inibidores , Técnicas In Vitro , Células Intersticiais de Cajal/citologia , Células Intersticiais de Cajal/efeitos dos fármacos , Células Intersticiais de Cajal/metabolismo , Fenômenos Mecânicos , Camundongos , Camundongos Endogâmicos , Contração Muscular/efeitos dos fármacos , Músculo Liso/irrigação sanguínea , Músculo Liso/citologia , Músculo Liso/inervação , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Concentração Osmolar , Plexo Submucoso/citologia , Plexo Submucoso/efeitos dos fármacos , Plexo Submucoso/metabolismo
15.
Dig Dis Sci ; 57(12): 3106-15, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22878915

RESUMO

BACKGROUND: In diabetes mellitus (DM), hyperglycemia promotes changes in biochemical mechanisms that induce oxidative stress. Oxidative stress has been closely linked to adverse consequences that affect the function of the gastrointestinal tract caused by injuries to the enteric nervous system (ENS) that in turn cause neurodegeneration and enteric glial loss. Therapeutic approaches have shown that diet supplementation with antioxidants, such as quercetin, reduce oxidative stress. AIMS: This work sought to evaluate neurons and enteric glial cells in the myenteric and submucosal plexuses of the duodenum in diabetic rats supplemented with quercetin. METHODS: The duodenum of 24 rats, including a control group (C), control quercetin supplementation group (CQ), diabetic group (D), and diabetic quercetin supplementation group (DQ), were used to investigate whole mounts of muscular and submucosal layers subjected to immunohistochemistry to detect vasoactive intestinal peptide in the myenteric layer and double-staining for HuC-D/neuronal nitric oxide synthase (nNOS) and HuC-D/S100. RESULTS: A reduction of the general neuronal population (HuC/D) was found in the myenteric and submucosal plexuses (p < 0.001) in the D and DQ groups. The nitrergic subpopulation (nNOS) decreased only in the myenteric plexus (p < 0.001), and glial cells decreased in both plexuses (p < 0.001) in the D and DQ groups. In diabetic rats, quercetin supplementation reduced neuronal and glial loss. Diabetes promoted an increase in the cell body area of both the general and nitrergic populations. Quercetin supplementation only prevented neuronal hypertrophy in the general population. CONCLUSION: Supplementation with quercetin eased the damage caused by diabetes, promoting a neuroprotective effect and reducing enteric glial loss in the duodenum.


Assuntos
Diabetes Mellitus Experimental/complicações , Duodeno/inervação , Sistema Nervoso Entérico/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Quercetina/farmacologia , Animais , Feminino , Masculino , Plexo Mientérico/citologia , Plexo Mientérico/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ratos , Ratos Wistar , Estreptozocina , Plexo Submucoso/citologia , Plexo Submucoso/efeitos dos fármacos
16.
Neurogastroenterol Motil ; 23(4): e165-70, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21223453

RESUMO

BACKGROUND: Leptin, one of the most prominent mediators released from adipocytes, influences neuronal activity in the central nervous system. The enteric nervous system (ENS) expresses leptin receptors but consequence of activation of these receptors on enteric neuron activity has not been systematically studied. An adipocyte-ENS axis is suggested by close apposition between enteric nerves and adipocytes. The aim of this study was to investigate the effects of leptin on guinea-pig submucous and myenteric neurons. METHODS: Using voltage sensitive dye imaging, we recorded neural responses to application of leptin (0.0625 nmol L(-1)) in myenteric and submucous neurons, nicotine (10 µmol L(-1)) served as a reference for neuronal excitation. Mucosal ion secretion and muscle activity were measured in vitro with Ussing and organ bath techniques, respectively. KEY RESULTS: Leptin induced spike discharge in 13.6% of submucous neurons and in 8.2% of myenteric neurons (1.1 ± 0.9 and 1.2 ± 1.0 Hz, respectively). Although there was an overlap of nicotine and leptin responses, 38.5% of submucous and 25% of myenteric neurons activated by leptin did not respond to nicotine. Leptin did not inhibit ongoing spike discharge or fast excitatory postsynaptic potentials. Leptin (0.0625 nmol L(-1)) did not affect mucosal secretion or muscle activity suggesting a subtle modulatory action of leptin at the level of the ENS. CONCLUSIONS & INFERENCES: Leptin activates submucous and myenteric neurons indicating relevance for adipocyte-ENS signaling. These results set the basis for further studies to reveal the functional correlate of the neural action of leptin in the ENS.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Sistema Nervoso Entérico/citologia , Leptina/farmacologia , Plexo Mientérico/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Plexo Submucoso/efeitos dos fármacos , Potenciais de Ação/fisiologia , Adipócitos/citologia , Adipócitos/fisiologia , Animais , Relação Dose-Resposta a Droga , Sistema Nervoso Entérico/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Estimulantes Ganglionares/farmacologia , Cobaias , Masculino , Modelos Animais , Plexo Mientérico/citologia , Plexo Mientérico/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Nicotina/farmacologia , Transdução de Sinais/fisiologia , Plexo Submucoso/citologia , Plexo Submucoso/fisiologia
17.
J Pharmacol Exp Ther ; 336(1): 178-87, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20876748

RESUMO

Macrolide antibiotics such as erythromycin (EM) and azithromycin (AZM) are beneficial in the treatment of mucus hypersecretion in inflammatory pulmonary diseases. Several indirect and direct mechanisms of action have been proposed. This study investigates the direct effect of macrolides on secretory function of isolated submucosal mucous gland cells (SMGCs). We hypothesize that macrolides inhibit the calcium influx necessary for evoked mucus secretion. To test this, we quantified mucin protein release using enzyme-linked immunosorbent assay, calcium-activated K(+) (K(Ca)), and calcium-activated Cl(-) (Cl(Ca)) currents. We measured nonselective cation current (NSCC) using whole-cell patch-clamp techniques; intracellular calcium concentration ([Ca(2+)](i)) was measured using fura-2 Ca(2+) imaging. We found that both EM and AZM are agonists at muscarinic receptors. EM (10 µM) evoked a small but significant increase in mucin release but inhibited the mucin release induced by subsequent acetylcholine (ACh) treatment. Both EM and AZM (10 µM) evoked K(Ca) and Cl(Ca) whole-cell currents, which were blocked by atropine. EM and AZM also accelerated the decay of inositol trisphosphate-induced K(Ca) and Cl(Ca) currents without changing the peak current amplitudes. Likewise, internal application of AZM (10 µM) enhanced the decay rate of ACh-induced K(Ca) and Cl(Ca) currents. EM (1-10 µM) and AZM (0.1-10 µM) slowly (over 25-30 min) inhibited thapsigargin (TG)-induced Ca(2+) entry when applied during the plateau phase of Ca(2+) entry but blunted TG-induced Ca(2+) entry by 70% after a 5-min pretreatment before initiating calcium entry. EM blocked TG-induced NSCC. We conclude that macrolide antibiotics are partial agonists at muscarinic receptors but inhibit stimulated mucus release by inhibiting calcium entry in SMGCs.


Assuntos
Cálcio/antagonistas & inibidores , Macrolídeos/farmacologia , Muco/efeitos dos fármacos , Muco/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Animais , Antibacterianos/farmacologia , Cálcio/metabolismo , Mucinas/antagonistas & inibidores , Mucinas/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Plexo Submucoso/efeitos dos fármacos , Plexo Submucoso/metabolismo , Suínos
18.
Am J Physiol Gastrointest Liver Physiol ; 298(3): G384-94, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20007849

RESUMO

Vasoactive intestinal peptide (VIP) immunoreactive neurons are important secretomotor neurons in the submucous plexus. They are the only submucosal neurons to receive inhibitory inputs and exhibit both noradrenergic and nonadrenergic inhibitory synaptic potentials (IPSPs). The former are mediated by alpha(2)-adrenoceptors, but the receptors mediating the latter have not been identified. We used standard intracellular recording, RT-PCR, and confocal microscopy to test whether 5-HT(1A), SST(1), and/or SST(2) receptors mediate nonadrenergic IPSPs in VIP submucosal neurons in guinea pig ileum in vitro. The specific 5-HT(1A) receptor antagonist WAY 100135 (1 microM) reduced the amplitude of IPSPs, an effect that persisted in the presence of the alpha(2)-adrenoceptor antagonist idazoxan (2 microM), suggesting that 5-HT might mediate a component of the IPSPs. Confocal microscopy revealed that there were many 5-HT-immunoreactive varicosities in close contact with VIP neurons. The specific SSTR(2) antagonist CYN 154806 (100 nM) and a specific SSTR(1) antagonist SRA 880 (3 microM) each reduced the amplitude of nonadrenergic IPSPs and hyperpolarizations evoked by somatostatin. In contrast with the other antagonists, CYN 154806 also reduced the durations of nonadrenergic IPSPs. Effects of WAY 100135 and CYN 154806 were additive. RT-PCR revealed gene transcripts for 5-HT(1A), SST(1), and SST(2) receptors in stripped submucous plexus preparations consistent with the pharmacological data. Although the involvement of other neurotransmitters or receptors cannot be excluded, we conclude that 5-HT(1A), SST(1), and SST(2) receptors mediate nonadrenergic IPSPs in the noncholinergic (VIP) secretomotor neurons. This study thus provides the tools to identify functions of enteric neural pathways that inhibit secretomotor reflexes.


Assuntos
Íleo/inervação , Potenciais Pós-Sinápticos Inibidores/fisiologia , Receptor 5-HT1A de Serotonina/genética , Receptores de Somatostatina/genética , Plexo Submucoso/fisiologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Expressão Gênica/genética , Cobaias , Idazoxano/farmacologia , Íleo/efeitos dos fármacos , Íleo/fisiologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/metabolismo , Plexo Mientérico/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Norepinefrina/farmacologia , Oligopeptídeos/farmacologia , Piperazinas/farmacologia , Quinolinas/farmacologia , Receptores de Somatostatina/antagonistas & inibidores , Serotonina/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina , Antagonistas do Receptor 5-HT1 de Serotonina , Agonistas do Receptor de Serotonina/farmacologia , Somatostatina/farmacologia , Plexo Submucoso/efeitos dos fármacos , Plexo Submucoso/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo
19.
Am J Physiol Gastrointest Liver Physiol ; 297(1): G135-43, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19389802

RESUMO

To test whether transient receptor potential channel vanilloid subfamily member-1 (TRPV1) mediates acid-induced inflammation in the esophagus, a tubular segment of esophageal mucosa was tied at both ends, forming a sac. The sac was filled with 0.01 N HCl (or Krebs buffer for control) and kept in oxygenated Krebs buffer at 37 degrees C. The medium around the sac (supernatant) was collected after 3 h. Supernatant of the HCl-filled sac abolished contraction of esophageal circular muscle strips in response to electric field stimulation. Contraction was similarly abolished by supernatant of mucosal sac filled with the TRPV1 agonist capsaicin (10(-6) M). These effects were reversed by the selective TRPV1 antagonist 5'-iodoresiniferatoxin (IRTX) and by the platelet-activating factor (PAF) receptor antagonist CV9388. Substance P and CGRP levels in mucosa and in supernatant increased in response to HCl, and these increases were abolished by IRTX and by tetrodotoxin (TTX) but not affected by CV9388, indicating that substance P and CGRP are neurally released and PAF independent. In contrast, the increase in PAF was blocked by IRTX but not by TTX. Presence of TRPV1 receptor was confirmed by RT-PCR and by Western blot analysis in whole mucosa and in esophageal epithelial cells enzymatically isolated and sorted by flow cytometry or immunoprecipitated with cytokeratin antibodies. In epithelial cells PAF increased in response to HCl, and the increase was abolished by IRTX. We conclude that HCl-induced activation of TRPV1 receptors in esophageal mucosa causes release of substance P and CGRP from neurons and release of PAF from epithelial cells.


Assuntos
Células Epiteliais/metabolismo , Esôfago/metabolismo , Ácido Clorídrico/metabolismo , Membrana Mucosa/metabolismo , Contração Muscular , Plexo Submucoso/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Capsaicina/farmacologia , Gatos , Diterpenos/farmacologia , Estimulação Elétrica , Células Epiteliais/efeitos dos fármacos , Esôfago/efeitos dos fármacos , Esôfago/inervação , Técnicas In Vitro , Masculino , Membrana Mucosa/efeitos dos fármacos , Membrana Mucosa/inervação , Contração Muscular/efeitos dos fármacos , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Estimulação Química , Plexo Submucoso/efeitos dos fármacos , Substância P/metabolismo , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Cátion TRPV/genética , Tetrodotoxina/farmacologia
20.
Neuroreport ; 20(3): 325-30, 2009 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-19190523

RESUMO

Serotonin (5-HT) plays a significant role in the regulation of intestinal secretion of water and electrolytes. The initial aim of this study was to use intracellular recording and specific antagonists to identify roles of 5-HT1A and 5-HT7 receptors of submucosal noncholinergic secretomotor neurons of guinea pig ileum, in vitro. However, it was found that the widely used 5-HT receptor antagonists NAN-190 (5-HT1A) and SB 269970 (5-HT7) both blocked alpha2-adrenoceptors, and hence depressed inhibitory synaptic potentials and hyperpolarizations evoked by noradrenaline, in these neurons. Both compounds enhanced neurally evoked contractions of the guinea pig vas deferens, an effect characteristic of blockade of alpha2-adrenoceptors. These results raise significant concerns about studies using NAN-190 and SB 269970 as specific antagonists of serotonin receptors.


Assuntos
Antagonistas de Receptores Adrenérgicos alfa 2 , Neurônios/efeitos dos fármacos , Fenóis/farmacologia , Piperazinas/farmacologia , Receptores de Serotonina/efeitos dos fármacos , Plexo Submucoso/efeitos dos fármacos , Sulfonamidas/farmacologia , Animais , Artefatos , Interações Medicamentosas/fisiologia , Feminino , Cobaias , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Músculo Liso/metabolismo , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Neurônios/metabolismo , Norepinefrina/antagonistas & inibidores , Norepinefrina/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Receptores de Serotonina/metabolismo , Antagonistas da Serotonina/farmacologia , Plexo Submucoso/metabolismo , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/inervação , Ducto Deferente/metabolismo
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