RESUMEN
AIM: To investigate whether glucagon-like peptide-2 (GLP-2) influences the neurally-induced responses in gastric strips from mice, since no data are available. METHODS: For functional experiments, gastric fundal strips were mounted in organ baths containing Krebs-Henseleit solution. Mechanical responses were recorded via force-displacement transducers, which were coupled to a polygraph for continuous recording of isometric tension. Electrical field stimulation (EFS) was applied via two platinum wire rings through which the preparation was threaded. The effects of GLP-2 (2 and 20 nmol/L) were evaluated on the neurally-induced contractile and relaxant responses elicited by EFS. Neuronal nitric oxide synthase (nNOS) enzyme was evaluated by immunohistochemistry. RESULTS: In the functional experiments, electrical field stimulation (EFS, 4-16 Hz) induced tetrodotoxin (TTX)-sensitive contractile responses, which were reduced in amplitude by GLP-2 (P < 0.05). In the presence of the nitric oxide (NO) synthesis inhibitor L-NNA, GLP-2 no longer influenced the neurally-evoked contractile responses (P > 0.05). The direct smooth muscle response to methacholine was not influenced by GLP-2 (P > 0.05). In the presence of guanethidine and carbachol, the addition of GLP-2 to the bath medium evoked TTX-sensitive relaxant responses that were unaffected by L-NNA (P > 0.05). EFS induced a fast NO-mediated relaxation, whose amplitude was enhanced in the presence of the hormone (P < 0.05). Immunohistochemical experiments showed a significant increase (P < 0.05) in nNOS immunoreactivity in the nerve structures after GLP-2 exposure. CONCLUSION: The results demonstrate that in gastric fundal strips, GLP-2 influences the amplitude of neurally-induced responses through the modulation of the nitrergic neurotransmission and increases nNOS expression.
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Fundus Gástrico/fisiología , Motilidad Gastrointestinal/fisiología , Péptido 2 Similar al Glucagón/fisiología , Músculo Liso/fisiología , Transmisión Sináptica/fisiología , Animales , Estimulación Eléctrica , Femenino , Fundus Gástrico/inervación , Ratones , Contracción Muscular/fisiología , Relajación Muscular/fisiología , Músculo Liso/inervación , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismoRESUMEN
OBJECTIVE: Impaired gastric accommodation is reported in patients with functional dyspepsia (FD). Previous findings in postinfectious patients with FD suggest that low-grade inflammation and dysfunction of nitrergic nerves play a role in impaired accommodation. To date, spontaneous animal models to study the relationship between these changes are lacking. We hypothesise that the normoglycaemic BioBreeding diabetes-prone (BB-DP) rat provides an animal model of inflammation-induced impaired gastric motor function. DESIGN: Control diabetes-resistant biobreeding, normoglycaemic and hyperglycaemic BB-DP rats were sacrificed at the age of 30, 70 and 220â days and gastric fundus tissue was harvested to study nitrergic motor control, inflammation and expression of neuronal isoform of nitric oxide synthase (nNOS) and inducible isoform of nitric oxide synthase (iNOS). Nutrient-induced changes in intragastric pressure (IGP) were measured in normoglycaemic BB-DP rats to study accommodation. RESULTS: No differences in nitrergic function and inflammation were observed between BB-DP and control rats at 30â days. The nitrergic component of the fundic muscle relaxation was reduced in BB-DP rats of 70 and 220â days. This was accompanied by a significant loss of nNOS proteins. IGP significantly increased during nutrient infusion in BB-DP rats of 220â days, indicating impaired accommodation. Infiltration of polymorphonuclear cells, increased myeloperoxidase activity and increased expression of iNOS was observed in the fundic mucosa and muscularis propria of 70-day-old and 220-day-old BB-DP rats. CONCLUSIONS: BB-DP rats of 220â days display altered fundic motor control and impaired accommodation, which is least partially explained by loss of nitrergic function. This may be related to inflammatory changes in the neuromuscular layer, suggesting that normoglycaemic BB-DP rats provide a spontaneous model for inflammation-induced impaired gastric accommodation.
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Modelos Animales de Enfermedad , Dispepsia/fisiopatología , Neuronas Nitrérgicas/fisiología , Ratas Endogámicas BB/fisiología , Estómago/fisiopatología , Animales , Biomarcadores/metabolismo , Western Blotting , Fundus Gástrico/inervación , Fundus Gástrico/metabolismo , Fundus Gástrico/fisiopatología , Mucosa Gástrica/metabolismo , Hiperglucemia/fisiopatología , Inmunohistoquímica , Neuronas Nitrérgicas/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estómago/inervaciónRESUMEN
Gastric hypersensitivity is one of the key contributors to the postprandial symptoms of epigastric pain/discomfort, satiety, and fullness in functional dyspepsia patients. Epidemiological studies found that adverse early-life experiences are risk factors for the development of gastric hypersensitivity. Preclinical studies found that neonatal colon inflammation elevates plasma norepinephrine (NE), which upregulates expression of nerve growth factor (NGF) in the muscularis externa of the gastric fundus. Our goal was to investigate the cellular mechanisms by which NE upregulates the expression of NGF in gastric hypersensitive (GHS) rats, which were subjected previously to neonatal colon inflammation. Neonatal colon inflammation upregulated NGF protein, but not mRNA, in the gastric fundus of GHS rats. Western blotting showed upregulation of p110γ of phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K), phosphoinositide-dependent kinase-1 (PDK1), pAKT(Ser473), and phosphorylated 4E-binding protein (p4E-BP1)(Thr70), suggesting AKT activation and enhanced NGF protein translation. AKT inhibitor MK-2206 blocked the upregulation of NGF in the fundus of GHS rats. Matrix metalloproteinase 9 (MMP-9), the major NGF-degrading protease, was suppressed, indicating that NGF degradation was impeded. Incubation of fundus muscularis externa with NE upregulated NGF by modulating the protein translation and degradation pathways. Yohimbine, an α2-adrenergic receptor antagonist, upregulated plasma NE and NGF expression by activating the protein translation and degradation pathways in naive rats. In contrast, a cocktail of adrenergic receptor antagonists suppressed the upregulation of NGF by blocking the activation of the protein translation and degradation pathways. Our findings provide evidence that the elevation of plasma NE induces NGF expression in the gastric fundus.
Asunto(s)
Dolor Abdominal/metabolismo , Colitis/metabolismo , Colon/metabolismo , Fundus Gástrico/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Norepinefrina/sangre , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/metabolismo , Dolor Abdominal/etiología , Dolor Abdominal/fisiopatología , Antagonistas Adrenérgicos/farmacología , Factores de Edad , Animales , Animales Recién Nacidos , Proteínas Portadoras/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Colitis/inducido químicamente , Colitis/fisiopatología , Colon/efectos de los fármacos , Colon/inervación , Colon/fisiopatología , Modelos Animales de Enfermedad , Fundus Gástrico/efectos de los fármacos , Fundus Gástrico/inervación , Fundus Gástrico/fisiopatología , Péptidos y Proteínas de Señalización Intracelular , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Estabilidad Proteica , Proteolisis , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-Dawley , Transducción de Señal , Ácido Trinitrobencenosulfónico , Regulación hacia ArribaRESUMEN
AIM: To investigate the relationship between neuronal nitric oxide synthase (nNOS) expression and the natriuretic peptide signaling pathway in the gastric fundus of streptozotocin (STZ)-induced diabetic mice. METHODS: Diabetic mice were induced by injection of STZ solution. Immunofluorescence labeling of HuC/D, nNOS and natriuretic peptide receptor-A, B, C (NPRs) in the gastric fundus (GF) was used to observe nNOS expression and whether NPRs exist on enteric neurons. The expression levels of nNOS and NPRs in the diabetic GF were examined by western blotting. An isometric force transducer recorded the electric field stimulation (EFS)-induced relaxation and contraction in the diabetic GF. An intracellular recording method assessed EFS-induced inhibitory junction potentials (IJP) on the GF. GF smooth muscles acquired from normal mice were incubated with different concentrations of the NPRs agonist C-type natriuretic peptide (CNP) for 24 h, after which their nNOS expressions were detected by western blotting. RESULTS: Eight weeks after injection, 43 diabetic mice were obtained from mouse models injected with STZ. Immunofluorescence indicated that the number of NOS neurons was significantly decreased and that nNOS expression was significantly downregulated in the diabetic GF. The results of physiological and electrophysiological assays showed that the EFS-induced relaxation that mainly caused by NO was significantly reduced, while the contraction was enhanced in the diabetic GF. EFS-induced IJP showed that L-NAME sensitive IJP in the diabetic GF was significantly reduced compared with control mice. However, both NPR-A and NPR-B were detected on enteric neurons, and their expression levels were upregulated in the diabetic GF. The nNOS expression level was downregulated dose-dependently in GF smooth muscle tissues exposed to CNP. CONCLUSION: These findings suggested that upregulation of the NPs signaling pathway may be involved in GF neuropathy caused by diabetes by decreasing nNOS expression.
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Diabetes Mellitus Experimental/enzimología , Sistema Nervioso Entérico/enzimología , Fundus Gástrico/inervación , Músculo Liso/inervación , Péptidos Natriuréticos/metabolismo , Neuronas Nitrérgicas/enzimología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/fisiopatología , Regulación hacia Abajo , Estimulación Eléctrica , Sistema Nervioso Entérico/fisiopatología , Masculino , Ratones Endogámicos ICR , Contracción Muscular , Óxido Nítrico/metabolismo , Receptores del Factor Natriurético Atrial/metabolismo , Transducción de Señal , Estreptozocina , Técnicas de Cultivo de Tejidos , Regulación hacia ArribaRESUMEN
Nitric oxide (NO) is a major inhibitory neurotransmitter in the gastrointestinal (GI) tract. Its main effector, NO-sensitive guanylyl cyclase (NO-GC), is expressed in several GI cell types, including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and fibroblast-like cells. Up to date, the interplay between neurons and these cells to initiate a nitrergic inhibitory junction potential (IJP) is unclear. Here, we investigate the origin of the nitrergic IJP in murine fundus and colon. IJPs were determined in fundus and colon SMC of mice lacking NO-GC globally (GCKO) and specifically in SMC (SM-GCKO), ICC (ICC-GCKO), and both SMC/ICC (SM/ICC-GCKO). Nitrergic IJP was abolished in ICC-GCKO fundus and reduced in SM-GCKO fundus. In the colon, the amplitude of nitrergic IJP was reduced in ICC-GCKO, whereas nitrergic IJP in SM-GCKO was reduced in duration. These results were corroborated by loss of the nitrergic IJP in global GCKO. In conclusion, our results prove the obligatory role of NO-GC in ICC for the initiation of an IJP. NO-GC in SMC appears to enhance the nitrergic IJP, resulting in a stronger and prolonged hyperpolarization in fundus and colon SMC, respectively. Thus NO-GC in both cell types is mandatory to induce a full nitrergic IJP. Our data from the colon clearly reveal the nitrergic IJP to be biphasic, resulting from individual inputs of ICC and SMC.
Asunto(s)
Colon/inervación , Fundus Gástrico/inervación , Células Intersticiales de Cajal/metabolismo , Inhibición Neural , Neuronas Nitrérgicas/metabolismo , Óxido Nítrico/metabolismo , Transmisión Sináptica , Animales , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Potenciales Postsinápticos Inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/metabolismo , Factores de TiempoRESUMEN
Nausea is the subjective unpleasant sensation that immediately precedes vomiting. Studies using barostats suggest that gastric fundus and lower esophageal sphincter (LES) relaxation precede vomiting. Unlike barostat, high-resolution manometry allows less invasive, detailed measurements of fundus pressure (FP) and axial movement of the gastroesophageal junction (GEJ). Nausea was induced in 12 healthy volunteers by a motion video and rated on a visual analog scale. FP was measured as the mean value of the five pressure channels that were clearly positioned below the LES. After intubation, a baseline (BL) recording of 15 min was obtained. This was followed by presentation of the motion video (at least 10 min, maximum 20 min) followed by 30 min recovery recording. Throughout the experiment we recorded autonomic nervous system (ANS) parameters [blood pressure, heart rate (HR), and cardiac vagal tone (CVT), which reflects efferent vagal activity]. Ten out of 12 subjects showed a drop in FP during peak nausea compared with BL (-4.0 ± 0.8 mmHg; P = 0.005), and 8/10 subjects showed a drop in LES pressure (-8.8 ± 2.5 mmHg; P = 0.04). Peak nausea preceded peak fundus and LES pressure drop. Nausea was associated with configuration changes at the GEJ such as LES shortening and esophageal lengthening. During nausea we observed a significantly increased HR and decreased CVT. In conclusion, nausea is associated with a drop in fundus and LES pressure, configuration changes at the GEJ as well as changes in the ANS activity such as an increased sympathetic tone (increased HR) and decreased parasympathetic tone (decreased CVT).
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Esfínter Esofágico Inferior/fisiopatología , Fundus Gástrico/fisiopatología , Manometría , Mareo por Movimiento/fisiopatología , Presión Sanguínea , Deglución , Esfínter Esofágico Inferior/inervación , Femenino , Fundus Gástrico/inervación , Reflujo Gastroesofágico/etiología , Reflujo Gastroesofágico/fisiopatología , Motilidad Gastrointestinal , Voluntarios Sanos , Frecuencia Cardíaca , Humanos , Intubación Gastrointestinal , Masculino , Mareo por Movimiento/etiología , Estimulación Luminosa , Presión , Salivación , Sistema Nervioso Simpático/fisiopatología , Factores de Tiempo , Nervio Vago/fisiopatología , Grabación en Video , Adulto JovenRESUMEN
The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction.
Asunto(s)
Fundus Gástrico/fisiología , Relajación Muscular , Músculo Liso/fisiología , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Óxido Nítrico/metabolismo , Pene/fisiología , Animales , Fundus Gástrico/efectos de los fármacos , Fundus Gástrico/inervación , Técnicas In Vitro , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Cadenas Pesadas de Miosina/deficiencia , Miosina Tipo V/deficiencia , NG-Nitroarginina Metil Éster/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Pene/efectos de los fármacos , Pene/inervaciónRESUMEN
Ca(2+) sensitization of contraction has typically been investigated by bathing muscles in solutions containing agonists. However, it is unknown whether bath-applied agonists and enteric neurotransmission activate similar Ca(2+) sensitization mechanisms. We investigated protein kinase C (PKC)-potentiated phosphatase inhibitor protein of 17 kDa (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation in murine gastric fundus muscles stimulated by bath-applied carbachol (CCh) or cholinergic motor neurotransmission. CCh increased MYPT1 phosphorylation at Thr696 (pT696) and Thr853 (pT853), CPI-17 at Thr38 (pT38), and myosin light chain at Ser19 (pS19). Electrical field stimulation (EFS) only increased pT38. In the presence of neostigmine, EFS increased pT38, pT853 and pS19. In fundus muscles of W/W(v) mice, EFS alone increased pT38 and pT853. Atropine blocked all contractions and all increases in pT696, pT853, pT38 and pS19. The Rho kinase (ROCK) inhibitor SAR1x blocked increases in pT853 and pT696. The PKC inhibitors Go6976 and Gf109203x or nicardipine blocked increases in pT38 and pT696. These findings suggest that cholinergic motor neurotransmission activates PKC-dependent CPI-17 phosphorylation. Bath-applied CCh recruits additional ROCK-dependent MYPT1 phosphorylation due to exposure of the agonist to a wider population of muscarinic receptors. Intramuscular interstitial cells of Cajal (ICC-IMs) and cholinesterases restrict ACh accessibility to a select population of muscarinic receptors, possibly only those expressed by ICC-IMs. These results provide the first biochemical evidence for focalized (or synaptic-like) neurotransmission, rather than diffuse 'volume' neurotransmission in a smooth muscle tissue. Furthermore, these findings demonstrate that bath application of contractile agonists to gastrointestinal smooth muscles does not mimic physiological responses to cholinergic neurotransmission.
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Calcio/metabolismo , Fundus Gástrico/fisiología , Transmisión Sináptica , Animales , Fibras Colinérgicas/efectos de los fármacos , Fibras Colinérgicas/fisiología , Estimulación Eléctrica , Fundus Gástrico/inervación , Fundus Gástrico/metabolismo , Células Intersticiales de Cajal/fisiología , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos C57BL , Antagonistas Muscarínicos/farmacología , Contracción Muscular , Proteínas Musculares/metabolismo , Músculo Liso/inervación , Músculo Liso/metabolismo , Músculo Liso/fisiología , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera , Neostigmina/farmacología , Fosfoproteínas/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Acute Helicobacter pylori infection produces hypochlorhydria. The decrease in acid facilitates survival of the bacterium and its colonization of the stomach. The present study was designed to identify the pathways in oxyntic mucosa by which acute H. pylori infection inhibits acid secretion. In rat fundic sheets in an Ussing chamber, perfusion of the luminal surface with H. pylori in spent broth (10(3)-10(8) cfu/ml) or spent broth alone (1:10(5) to 1:10(0) final dilution) caused a concentration-dependent increase in somatostatin (SST; maximal: 200 ± 20 and 194 ± 9% above basal; P < 0.001) and decrease in histamine secretion (maximal: 45 ± 5 and 48 ± 2% below basal; P < 0.001); the latter was abolished by SST antibody, implying that changes in histamine secretion reflected changes in SST secretion. Both responses were abolished by the axonal blocker tetrodotoxin (TTX), the sensory neurotoxin capsaicin, or the CGRP antagonist CGRP8-37, implying that the reciprocal changes in SST and histamine secretion were due to release of CGRP from sensory neurons. In isolated rabbit oxyntic glands, H. pylori inhibited basal and histamine-stimulated acid secretion in a concentration-dependent manner; the responses were not affected by TTX or SST antibody, implying that H. pylori can directly inhibit parietal cell function. In conclusion, acute administration of H. pylori is capable of inhibiting acid secretion directly as well as indirectly by activating intramural CGRP sensory neurons coupled to stimulation of SST and inhibition of histamine secretion. Activation of neural pathways provides one explanation as to how initial patchy colonization of the superficial gastric mucosa by H. pylori can acutely inhibit acid secretion.
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Aclorhidria/microbiología , Péptido Relacionado con Gen de Calcitonina/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Células Receptoras Sensoriales/metabolismo , Somatostatina/metabolismo , Aclorhidria/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/antagonistas & inhibidores , Péptido Relacionado con Gen de Calcitonina/farmacología , Modelos Animales de Enfermedad , Ácido Gástrico/metabolismo , Fundus Gástrico/inervación , Fundus Gástrico/metabolismo , Fundus Gástrico/microbiología , Mucosa Gástrica/inervación , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Células HeLa , Infecciones por Helicobacter/microbiología , Histamina/metabolismo , Humanos , Células Parietales Gástricas/metabolismo , Células Parietales Gástricas/microbiología , Fragmentos de Péptidos/farmacología , Conejos , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/microbiología , Bloqueadores de los Canales de Sodio/farmacología , Somatostatina/farmacología , Tetrodotoxina/farmacologíaRESUMEN
Glucagon-like peptide-2 (GLP-2) influences different aspects of the gastrointestinal function, including epithelial growth, digestion, absorption, motility, and blood flow. Intraluminal pressure from isolated mouse stomach was recorded to investigate whether GLP-2 affects gastric tone and to analyze its mechanism of action. Regional differences between diverse parts of the stomach were also examined using circular muscular strips from fundus and antrum. In the whole stomach, GLP-2 (0.3-100 nM) produced concentration-dependent relaxation with a maximum that was about 75% of relaxation to 1 microM isoproterenol (IC50=2.5 nM). This effect was virtually abolished by desensitization of GLP-2 receptors or by alpha-chymotrypsin. The relaxant response to GLP-2 was not affected by tetrodotoxin, a blocker of neuronal voltage-dependent Na+ channels, but it was significantly reduced by omega-conotoxin GVIA, a blocker of neuronal N-type voltage-operated Ca2+ channels. Nomega-nitro-L-arginine methyl ester, a blocker of nitric oxide synthase, or apamin, a blocker of Ca2+-dependent potassium channels, failed to affect the gastric response to the peptide. However, the relaxation was significantly antagonized by [Lys1,Pro2,5,Arg3,4,Tyr6]VIP7-28, a vasoactive intestinal peptide (VIP) receptor antagonist (GLP-2 maximum effect=45% of relaxation to 1 microM isoproterenol), and virtually abolished by desensitization of the VIP receptors. GLP-2 induced concentration-dependent relaxation in carbachol-precontracted fundic strips but not in antral strips. These results provide the first experimental evidence that GLP-2 is able to induce gastric relaxation acting peripherally on the mouse stomach. The effect appears to be mediated by prejunctional neural release of VIP and confined to fundic region.
Asunto(s)
Sistema Nervioso Entérico/fisiología , Vaciamiento Gástrico/fisiología , Fundus Gástrico/fisiología , Péptido 2 Similar al Glucagón/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Animales , Quimotripsina/farmacología , Inhibidores Enzimáticos/farmacología , Vaciamiento Gástrico/efectos de los fármacos , Fundus Gástrico/inervación , Péptido 2 Similar al Glucagón/farmacología , Técnicas In Vitro , Isoproterenol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , NG-Nitroarginina Metil Éster/farmacología , Antro Pilórico/inervación , Antro Pilórico/fisiología , Bloqueadores de los Canales de Sodio/farmacología , Simpatomiméticos/farmacología , Tetrodotoxina/farmacologíaRESUMEN
BACKGROUND & AIMS: Ghrelin is an orexigenic peptide with gastroprokinetic effects. Mice with streptozotocin (STZ)-induced diabetes exhibit hyperphagia, altered gastric emptying, and increased plasma ghrelin levels. We investigated the causative role of ghrelin herein by comparing changes in ghrelin receptor knockout (growth hormone secretagogue receptor [GHS-R](-/-)) and wild-type (GHS-R(+/+)) mice with STZ-induced diabetes. METHODS: Gastric emptying was measured with the [(13)C]octanoic acid breath test. The messenger RNA (mRNA) expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin was quantified by real-time reverse-transcription polymerase chain reaction. Neural contractions were elicited by electrical field stimulation in fundic smooth muscle strips. RESULTS: Diabetes increased plasma ghrelin levels to a similar extent in both genotypes. Hyperphagia was more pronounced in GHS-R(+/+) than in GHS-R(-/-) mice between days 12 and 21. Increases in NPY and AgRP mRNA expression were less pronounced in diabetic GHS-R(-/-) than in GHS-R(+/+) mice from day 15 on, whereas decreases in proopiomelanocortin mRNA levels were similar in both genotypes. Gastric emptying was accelerated to a similar extent in both genotypes, starting on day 16. In fundic smooth muscle strips of diabetic GHS-R(+/+) and GHS-R(-/-) mice, neuronal relaxations were reduced, whereas contractions were increased; this increase was related to an increased affinity of muscarinic and tachykinergic receptors. CONCLUSIONS: Diabetic hyperphagia is regulated by central mechanisms in which the ghrelin-signaling pathway affects the expression of NPY and AgRP in the hypothalamus. The acceleration of gastric emptying, which is not affected by ghrelin signaling, is not the cause of diabetic hyperphagia and probably involves local contractility changes in the fundus.
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Diabetes Mellitus Experimental/fisiopatología , Vaciamiento Gástrico/fisiología , Ghrelina/sangre , Hiperfagia/fisiopatología , Receptores de Ghrelina/genética , Acetilcolina/farmacología , Proteína Relacionada con Agouti/genética , Animales , Glucemia/metabolismo , Peso Corporal/fisiología , Colinérgicos/farmacología , Diabetes Mellitus Experimental/metabolismo , Ingestión de Alimentos/fisiología , Fundus Gástrico/inervación , Fundus Gástrico/fisiología , Ghrelina/genética , Hiperfagia/metabolismo , Hipotálamo/fisiología , Masculino , Ratones , Ratones Noqueados , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Neuropéptido Y/genética , Neurotransmisores/farmacología , Proopiomelanocortina/genética , ARN Mensajero/metabolismo , Receptores de Ghrelina/metabolismo , Sustancia P/farmacologíaRESUMEN
This study investigated the possible interaction between the heme oxygenase (HO)/biliverdin reductase (BVR) and nitric oxide synthase (NOS) pathway in murine gastric fundus and jejunum, since previous studies have shown that both HO-2 and BVR are expressed in interstitial cells of Cajal (ICCs) and co-localized with neuronal NOS in a large proportion of myenteric neurons along the gastrointestinal tract. Neither HO inhibition by chromium mesoporphyrin (CrMP) nor co-incubation with CO or biliverdin/bilirubin affected nitrergic neurotransmission - i.e. relaxations induced by non-adrenergic non-cholinergic (NANC) nerve stimulation or exogenous NO - under normal physiological conditions. However, biliverdin/bilirubin reversed the inhibitory effect of the superoxide generator LY83583 on exogenous NO-induced relaxations in both tissues. When gastric fundus muscle strips were depleted of the endogenous antioxidant Cu/Zn superoxide dismutase (SOD) by the Cu-chelator DETCA, electrically induced NANC relaxations were also affected by LY82583; however, biliverdin/bilirubin could not substitute for the loss of Cu/Zn SOD when this specific antioxidant enzyme was depleted. In jejunal muscle strips, the combination DETCA plus LY83583 nearly abolished contractile phasic activity and, hence, did not allow studying nitrergic relaxation in these experimental conditions. In conclusion, this study does not establish a role for HO/CO in inhibitory NANC neurotransmission in murine gastric fundus and jejunum under normal physiological conditions. However, the antioxidants biliverdin/bilirubin might play an important role in the protection of the nitrergic neurotransmitter against oxidative stress.
Asunto(s)
Fundus Gástrico/inervación , Hemo Oxigenasa (Desciclizante)/fisiología , Yeyuno/inervación , Óxido Nítrico Sintasa/fisiología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/fisiología , Transmisión Sináptica , Aminoquinolinas/farmacología , Animales , Biliverdina/farmacología , Monóxido de Carbono/farmacología , Estimulación Eléctrica , Estradiol/análogos & derivados , Estradiol/farmacología , Masculino , Ratones , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/fisiología , Compuestos Organometálicos/farmacología , Transducción de SeñalRESUMEN
Nicotine acts as an agonist of nicotinic acetylcholine receptors. Nicotinic acetylcholine receptors play a role in the modulation of neurotransmitter release in both the central and the peripheral nervous system. Moderate reactive oxygen species levels modulate the regulation of physiological functions e.g. neurotransmitter release. Previously in rabbit gastric fundus we demonstrated that nicotine transiently increased neurogenic contraction induced by electrical field stimulation (EFS). In this study we aimed to investigate the effects of hydrogen peroxide (H2O2), antioxidizing enzymes catalase and superoxide dismutase (SOD) on nicotine induced increases at cholinergic neurotransmission in rabbit gastric fundus. Although H2O2 did not alter nicotine induced transient neurogenic contractions at concentrations of 10(-6) and 10(-5) M, at high concentration (10(-4) M) H2O2 inhibited nicotine induced increases. Catalase (500 units/ml), enhanced the effect of nicotine but did not alter nicotine induced transient neurogenic contractions at the concentrations of 100 and 250 units/ml. SOD (75,150 and 225 units/ml) did not alter nicotine induced transient neurogenic contractions. In conclusion, at high concentration H2O2 (10(-4) M) inhibited nicotine's transient ability to augment neurogenic contractions and catalase (500 units/ml) enhanced the effect of nicotine.
Asunto(s)
Antioxidantes/farmacología , Catalasa/farmacología , Peróxido de Hidrógeno/farmacología , Músculo Liso/efectos de los fármacos , Músculo Liso/inervación , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Superóxido Dismutasa/farmacología , Animales , Atropina/farmacología , Estimulación Eléctrica , Fundus Gástrico/efectos de los fármacos , Fundus Gástrico/inervación , Técnicas In Vitro , Antagonistas Muscarínicos/farmacología , Contracción Muscular/efectos de los fármacos , Sistema Nervioso Parasimpático/efectos de los fármacos , Conejos , Bloqueadores de los Canales de Sodio/farmacología , Transmisión Sináptica/efectos de los fármacosRESUMEN
The sphincter mechanism at the esophagogastric junction includes smooth muscle of the lower esophagus and skeletal muscle of the crural diaphragm (CD). Smooth muscle is known to be under the control of the dorsal motor nucleus of the vagus (DMV), while central nervous system (CNS) control of the CD is unknown. The main purposes of our study were to determine the CNS site that controls the CD and whether simultaneous changes in lower esophageal sphincter (LES) pressure and CD activity occur when this site is activated. Experiments were performed on anesthetized male ferrets whose LES pressure, CD activity, and fundus tone were monitored. To activate DMV neurons, L-glutamate was microinjected unilaterally into the DMV at three areas: intermediate, rostral, and caudal. Stimulation of the intermediate DMV decreased CD activity (-4.8 +/- 0.1 bursts/min and -0.3 +/- 0.01 mV) and LES pressure (-13.2 +/- 2.0 mmHg; n = 9). Stimulation of this brain site also produced an increase in fundus tone. Stimulation of the rostral DMV elicited increases in the activity of all three target organs (n = 5). Stimulation of the caudal DMV had no effect on the CD but did decrease both LES pressure and fundus tone (n = 5). All changes in LES pressure, fundus tone, and some DMV-induced changes in CD activity (i.e., bursts/min) were prevented by ipsilateral vagotomy. Our data indicate that simultaneous changes in activity of esophagogastric sphincters and fundus tone occur from rostral and intermediate areas of the DMV and that these changes are largely mediated by efferent vagus nerves.
Asunto(s)
Diafragma/fisiología , Esfínter Esofágico Inferior/fisiología , Fundus Gástrico/fisiología , Nervio Vago/fisiología , Animales , Diafragma/inervación , Inhibidores Enzimáticos/farmacología , Esfínter Esofágico Inferior/inervación , Hurones , Fundus Gástrico/inervación , Ácido Glutámico/farmacología , Masculino , Microinyecciones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , NG-Nitroarginina Metil Éster/farmacología , Nervio Vago/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
To assess whether afferent vagal intramuscular arrays (IMAs), putative gastrointestinal mechanoreceptors, form contacts with interstitial cells of Cajal of the intramuscular type (ICC-IM) and to describe any such contacts, electron microscopic analyses were performed on the external muscle layers of the fundus containing dextran-labelled diaminobenzidin (DAB)-stained IMAs. Special staining and embedding techniques were developed to preserve ultrastructural features. Within the muscle layers, IMA varicosities were observed in nerve bundles traversing major septa without contact with ICC-IM, contacting unlabelled neurites and glial cells. IMA varicosities were encountered in minor septa in contact with ICC-IM which were not necessarily in close contact with muscle cells. In addition, IMA varicosities were observed within muscle bundles in close contact with ICC-IM which were in gap junction contact with muscle cells. IMAs formed varicosities containing predominantly small agranular vesicles, occasionally large granular vesicles and prejunctional thickenings in apposition to ICC-IM processes, indicating communication between ICC and IMA via synapse-like contacts. Taken together, these different morphological features are consistent with a hypothesized mechanoreceptor role for IMA-ICC complexes. Intraganglionic laminar ending varicosities contacted neuronal somata and dendrites in the myenteric plexus of the fundus, but no contacts with ICC associated with Auerbach's plexus were encountered.
Asunto(s)
Cuerpos Enrollados/fisiología , Fundus Gástrico/fisiología , Mecanorreceptores/fisiología , Nervio Vago/fisiología , Vías Aferentes/fisiología , Animales , Fundus Gástrico/inervación , Masculino , Músculo Liso/citología , Músculo Liso/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
The motility of the gastrointestinal tract is generated by smooth muscle cells and is controlled to a large extent by an intrinsic neural network. A gap of approximately 200 nm usually separates nerve varicosities from smooth muscle cells, which suggests that direct innervation of the smooth muscle by synapses does not occur. Enteric nerves do make synapse-like contact with proposed regulatory cells, the interstitial cells of Cajal (ICC), which in turn may be in gap junction contact with smooth muscle cells. The role played by ICC in enteric innervation is controversial. Experimental evidence has been presented in vitro for the hypothesis that nitrergic inhibitory innervation is strongly reduced in the absence of ICC. However, in vivo data appear to dispute that. The present report provides evidence that explains the discrepancy between in vivo and in vitro data and provides evidence that inhibitory neurotransmitters can reach smooth muscle cells without hindrance when ICC are absent. The fundic musculature shows increased responses to substance P-mediated innervation and shows marked spontaneous activity, which is consistent with increased muscle excitability.
Asunto(s)
Músculo Liso/fisiología , Óxido Nítrico/fisiología , Estómago/citología , Estómago/fisiología , Adrenérgicos/farmacología , Animales , Atropina/farmacología , Estimulación Eléctrica , Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/efectos de los fármacos , Sistema Nervioso Entérico/fisiología , Inhibidores Enzimáticos/farmacología , Fundus Gástrico/inervación , Fundus Gástrico/fisiología , Guanetidina/farmacología , Inmunohistoquímica , Ratones , Microscopía Electrónica , Agonistas Muscarínicos/farmacología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/inervación , Músculo Liso/ultraestructura , Neurotransmisores/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacología , Ratas , Estómago/ultraestructuraRESUMEN
The wall of the gastrointestinal tract presents extensive plexuses of nerve fibres and neuronal cell bodies responsible for the modulation of the rhythmic gastrointestinal peristaltic activities, among other functions. One of the most developed ganglionated plexuses of the gastrointestinal tract is the Myenteric plexus located between the inner circular layer and outer longitudinal layer of the smooth muscle tunica. The musculature of fundus, body and pyloric parts of stomach are differently disposed and they perform different functions. Thus the present study was conducted to study the myenteric plexus of all parts of stomach by counting the number of collections of neurons, number of neurons in each collection, diameter and area of the neurons of the plexus. The stomach walls of 1 cm in size were taken from 5 cadavers of medical post mortem cases from Postgraduate Institute of Medical Sciences and Research, Chandigarh and were processed for paraffin sections. 5 and 10 micro thick sections were stained with haematoxylin and Eosin and examined under light microscope. Randomly selected sections were photomicrographed using digital camera and morphometrical analysis was done using Image-Pro Express software. Number of collections of neurons was maximum in fundus with an average of 4.521 and each collection on an average contain 5.27 neurons ranging from 1-31, while body had 3.292 collections containing 1-19 neurons (mean: 3.198), pylorus had 3.883 collections of neurons which contained 1-16 neurons (mean: 4.411). The neurons were classified as small, medium and large according to the size of the area of their cell bodies. In this way, 11.3% neurons were found to be small, 69.5% medium and 19.1% large in fundus, 8.7% small, 80.6% medium and 11.2% large in body and 11.1% small, 74.3% medium and 14.5% large in pylorus.
Asunto(s)
Sistema Nervioso Autónomo/anatomía & histología , Fundus Gástrico/inervación , Músculo Liso/inervación , Plexo Mientérico/anatomía & histología , Píloro/inervación , Estómago/inervación , Ganglios , Humanos , Músculo Liso/fisiología , Plexo Mientérico/fisiología , Proyectos Piloto , Estómago/anatomía & histologíaRESUMEN
1 This study was undertaken to analyse the involvement of ATP in non-adrenergic non- cholinergic (NANC) relaxation and possible interplay between nitrergic and purinergic systems in rat gastric fundus. 2 Experiments were performed in vitro on strips of longitudinal muscle from rat gastric fundus, recording the mechanical activity as changes in isometric force. In addition, NO release induced by different experimental conditions was assayed. 3 Under NANC conditions in serotonin-precontracted strips, electrical field stimulation (EFS) elicited a tetrodotoxin (TTX)-sensitive relaxation accompanied by nitric oxide (NO) release. This effect was antagonized by pretreatment with the NO synthase antagonist Nomega-nitro-L-arginine (L-NA) or by desensitization of purinergic receptors. Purinergic desensitization was also able to further antagonize the residual EFS-induced relaxation remaining after L-NA treatment. Exogenously applied NO [delivered as sodium nitroprusside (SNP)] or ATP (and related purines) induced concentration-dependent, TTX-insensitive relaxant responses. ATP also induced the release of NO. A reduction in the responses to ATP was observed in the presence of L-NA. In contrast, SNP-induced relaxation remained unchanged after desensitization of purinergic receptors. Finally, apamin, a blocker of the small conductance Ca2+ -dependent K+ channels, reduced the amplitude of the muscular relaxation evoked by either EFS, ATP or SNP. 4 In conclusion, this study provides evidence that in rat gastric fundus, ATP is one of the inhibitory transmitters released from NANC intramural neurones acting directly on the muscle, through receptors coupled to apamin-sensitive Ca2+ -dependent K+ channels and, indirectly, through the stimulation of NO production.
Asunto(s)
Fundus Gástrico/fisiología , Músculo Liso/fisiología , Neuronas Nitrérgicas/fisiología , Receptores Purinérgicos/fisiología , Adenosina/farmacología , Adenosina Difosfato/farmacología , Adenosina Monofosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , Apamina/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Femenino , Fundus Gástrico/efectos de los fármacos , Fundus Gástrico/inervación , Técnicas In Vitro , Masculino , Relajación Muscular/efectos de los fármacos , Relajación Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/inervación , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacología , Nitroprusiato/farmacología , Ratas , Ratas Wistar , Tetrodotoxina/farmacologíaRESUMEN
We reported pharmacological data suggesting that stimulation of the vago-vagal reflex activates noradrenergic neurons in the hindbrain that inhibit dorsal motor nucleus of the vagus (DMV) neurons projecting to the fundus, but not to the antrum [Ferreira Jr., M., Sahibzada, N., Shi, M., Panico, W., Neidringhaus, M., Wasserman, A., Kellar, K.J., Verbalis, J., Gillis, R.A., 2002. CNS site of action and brainstem circuitry responsible for the intravenous effects of nicotine on gastric tone. J. Neurosci. 22, 2764-2779.]. The purpose of this study was to use an ultrastructural approach to test the hypothesis that noradrenergic terminals form synapses with DMV fundus-projecting neurons, but not with DMV antrum-projecting neurons. A retrograde tracer, CTbeta-HRP, was injected into the gastric smooth muscle of either the fundus or the antrum of rats. Animals were re-anesthetized 48 h later and perfusion-fixed with acrolein and paraformaldehyde. Brainstems were processed histochemically for CTbeta-HRP, and immunocytochemically for either DbetaH or PNMT by dual-labeling electron microscopic methods. Most cell bodies and dendrites of neurons that were retrogradely labeled from the stomach occurred at the level of the area postrema. Examination of 482 synapses on 238 neurons that projected to the fundus revealed that 17.4+/-2.7% (n=4) of synaptic contacts were with DbetaH-IR terminals. Of 165 fundus-projecting neurons, 4.4+/-1.5% (n=4) formed synaptic contacts with PNMT-IR terminals. In contrast, the examination of 384 synapses on 223 antrum-projecting neurons revealed no synaptic contact with DbetaH-IR terminals. These data provide proof that norepinephrine containing nerve terminals synapse with DMV fundus-projecting neurons but not with DMV antrum-projecting neurons. These data also suggest that brainstem circuitry controlling the fundus differs from circuitry controlling the antrum.
Asunto(s)
Fundus Gástrico/inervación , Norepinefrina/metabolismo , Rombencéfalo/ultraestructura , Nervio Vago/ultraestructura , Aferentes Viscerales/ultraestructura , Animales , Área Postrema/fisiología , Área Postrema/ultraestructura , Vías Autónomas/fisiología , Vías Autónomas/ultraestructura , Comunicación Celular/fisiología , Toxina del Cólera , Dendritas/fisiología , Dendritas/ultraestructura , Dopamina beta-Hidroxilasa/análisis , Dopamina beta-Hidroxilasa/metabolismo , Fundus Gástrico/fisiología , Peroxidasa de Rábano Silvestre , Inmunohistoquímica , Masculino , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Feniletanolamina N-Metiltransferasa/análisis , Feniletanolamina N-Metiltransferasa/metabolismo , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Rombencéfalo/fisiología , Sistema Nervioso Simpático/fisiología , Sistema Nervioso Simpático/ultraestructura , Transmisión Sináptica/fisiología , Nervio Vago/fisiología , Aferentes Viscerales/fisiologíaRESUMEN
Interstitial cells of Cajal (ICC) within the gastrointestinal (GI) tract play a critical role in the generation of electrical slow waves and as mediators of enteric motor neurotransmission. Kit immunohistochemistry has proven to be a reliable method to identify the location of these cells within the tunica muscularis and to provide information on how the distribution and density of these cells change in a variety of GI motility disorders. Because of the labile nature of Kit or its detection, ultrastructural immunocytochemistry using conventional chemical fixation methods has been difficult. We describe a novel in vivo technique to label ICC within GI tissues. Using antibodies directed against the extracellular domain of the Kit receptor, we have been able to live-label the stomach with Kit while the animal is under anaesthesia and the organ is still receiving normal blood supply. This approach provided optimum maintenance of ultrastructural features with significant binding of antibody to the Kit receptor. The loss of ICC in many human motility disorders suggests exciting new hypotheses for their aetiology. This method will prove useful to investigate the ultrastructural changes that occur in ICC networks in animal models of motility disorders that are associated with the loss of these cells.