RESUMEN
The enteroendocrine profile and distribution patterns of the taste signaling molecules, α-gustducin (Gαgust) and α-transducin (Gαtran) protein subunits, were studied in the gastrointestinal (GI) tract of the chicken (Gallus domesticus) using double labeling immunohistochemistry. Gαtran or Gαgust immunoreactivity was observed in enteroendocrine cells (EEC) expressing different peptides throughout the entire GI tract with different density. In the proventriculus tubular gland, Gαtran or Gαgust/gastrin (GAS) immunoreactive (-IR) cells were more abundant than Gαtran/or Gαgust containing glucagon-like peptide-1 (GLP-1) or peptide YY (PYY), whereas only few Gαtran or Gαgust cells co-stored ghrelin (GHR) or 5-hydroxytryptamine (5-HT). In the pyloric mucosa, many Gαtran or Gαgust-IR cells co-expressed GAS or GHR, with less Gαtran or Gαgust cells containing GLP-1, PYY, or 5-HT. In the small intestine, a considerable subset of Gαtran or Gαgust-IR cells co-expressed 5-HT in the villi of the duodenum and ileum, PYY in the villi of the jejunum, CCK or GLP-1 in the villi of the ileum, and GHR in the duodenum crypts. In the large intestine, many Gαtran or Gαgust-IR cells contained 5-HT or GLP-1 in the villi of the rectum, whereas some Gαtran/Gαgust-IR cells co-expressed PYY- or CCK-, and few Gαtran/Gαgust-IR cells were positive for GHR-IR. In the cecum, several Gαtran or Gαgust-IR cells were IR for 5-HT. Finally, many Gαtran/Gαgust cells containing 5-HT were observed in the villi and crypts of the cloaca, whereas there were few Gαtran or Gαgust/CCK-IR cells. The demonstration that Gα-subunits are expressed in the chicken GI enteroendocrine system supports the involvement of taste signaling machinery in the chicken chemosensing processes.
Asunto(s)
Peso Corporal/fisiología , Pollos/fisiología , Células Enteroendocrinas/citología , Tracto Gastrointestinal/citología , Saciedad/fisiología , Transducina/metabolismo , Animales , Aminas Biogénicas/metabolismo , Péptidos/metabolismo , FenotipoRESUMEN
BACKGROUND: Chronic constipation (CC) is a common and severe gastrointestinal complaint in Parkinson's disease (PD), but its pathogenesis remains poorly understood. This study evaluated functionally distinct submucosal neurons in relation to colonic motility and anorectal function in PD patients with constipation (PD/CC) vs both CC and controls. METHODS: Twenty-nine PD/CC and 10 Rome III-defined CC patients were enrolled. Twenty asymptomatic age-sex matched subjects served as controls. Colonic transit time measurement and conventional anorectal manometry were evaluated in PD/CC and CC patients. Colonoscopy was performed in all three groups. Colonic submucosal whole mounts from PD/CC, CC, and controls were processed for immunohistochemistry with antibodies for vasoactive intestinal polypeptide (VIP) and peripheral choline acetyltransferase, markers for functionally distinct submucosal neurons. The mRNA expression of VIP and its receptors were also assessed. KEY RESULTS: Four subgroups of PD/CC patients were identified: delayed colonic transit plus altered anorectal manometry (65%); delayed colonic transit (13%); altered manometric pattern (13%); and no transit and manometric impairment (9%). There were no differences in the number of neurons/ganglion between PD/CC vs CC or vs controls. A reduced number of submucosal neurons containing VIP immunoreactivity was found in PD/CC vs controls (P<.05). VIP, VIPR1, and VIPR2 mRNA expression was significantly reduced in PD/CC vs CC and controls (P<.05). CONCLUSIONS AND INFERENCES: Colonic motor and rectal sensory functions are impaired in most PD/CC patients. These abnormalities are associated with a decreased VIP expression in submucosal neurons. Both sensory-motor abnormalities and neurally mediated motor and secretory mechanisms are likely to contribute to PD/CC pathophysiology.
Asunto(s)
Estreñimiento/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Plexo Submucoso/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neuronas Colinérgicas/metabolismo , Enfermedad Crónica , Estreñimiento/complicaciones , Estreñimiento/fisiopatología , Regulación hacia Abajo , Femenino , Tránsito Gastrointestinal , Humanos , Masculino , Manometría , Persona de Mediana Edad , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/fisiopatología , ARN Mensajero/metabolismo , Enfermedades del Recto/complicaciones , Enfermedades del Recto/metabolismo , Enfermedades del Recto/fisiopatologíaRESUMEN
The expression and distribution patterns of the taste signaling molecules, α-gustducin (Gαgust) and α-transducin (Gαtran) G-protein subunits, were studied in the gastrointestinal tract of the chicken (Gallus domesticus) using the immunohistochemical method. Gαgust and Gαtran immunoreactive (-IR) cells were observed in the mucosal layer of all examined segments, except the esophagus, crop, and the saccus cranialis of the gizzard. The highest numbers of Gαgust and Gαtran-IR cells were found in the proventriculus glands and along the villi of the pyloric, duodenum, and rectal mucosa. Gαgust and Gαtran-IR cells located in the villi of the jejunum, ileum, and cloaca were much less numerous, while only a few Gαgust and Gαtran-IR cells were detected in the mucosa of the proventriculus and cecum. In the crypts, IR cells were observed in the small and large intestine as well as in the cloaca. Gαgust and Gαtran-IR cells displayed elongated ("bottle-" or "pear-like") or rounded shape. The demonstration of Gαgust and Gαtran expression provides evidence for taste receptor mediated mucosal chemosensitivity in the chicken gastrointestinal tract.
Asunto(s)
Proteínas Aviares/genética , Pollos/fisiología , Transducción de Señal , Transducina/genética , Animales , Proteínas Aviares/metabolismo , Pollos/genética , Tracto Gastrointestinal/fisiología , Expresión Génica , Masculino , Especificidad de Órganos , Gusto , Transducina/metabolismoRESUMEN
BACKGROUND: Specialized endoderm-derived epithelial cells, that is, enteroendocrine cells (EECs), are widely distributed throughout the gastrointestinal (GI) tract. Enteroendocrine cells form the largest endocrine organ in the body and play a key role in the control of GI secretion and motility, the regulation of food intake, postprandial glucose levels and metabolism. EECs sense luminal content and release signaling molecules that can enter the circulation to act as classic hormones on distant targets, act locally on neighboring cells and on distinct neuronal pathways including enteric and extrinsic neurons. Recent studies have shed light on EEC sensory transmission by showing direct connections between EECs and the nervous system via axon-like processes that form a well-defined neuroepithelial circuits through which EECs can directly communicate with the neurons innervating the GI tract to initiate appropriate functional responses. PURPOSE: This review will highlight the role played by the EECs in the complex and integrated sensory information responses, and discuss the new findings regarding EECs in the brain-gut axis bidirectional communication.
Asunto(s)
Encéfalo/fisiología , Comunicación Celular/fisiología , Células Enteroendocrinas/fisiología , Tracto Gastrointestinal/fisiología , Encéfalo/citología , Ingestión de Alimentos/fisiología , Hormonas Gastrointestinales/fisiología , Tracto Gastrointestinal/citología , Humanos , Vías Nerviosas/citología , Vías Nerviosas/fisiologíaRESUMEN
BACKGROUND: µ opioid receptors (µORs) are expressed by neurons and inflammatory cells, and mediate immune response. We tested whether activation of peripheral µORs ameliorates the acute and delayed phase of colitis. METHODS: C57BL/6J mice were treated with 3% dextran sodium sulfate (DSS) in water, 5 days with or without the peripherally acting µOR agonist, [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin (DAMGO) or with DAMGO+µOR antagonist at day 2-5, then euthanized. Other mice received DSS followed by water for 4 weeks, or DSS with DAMGO starting at day 2 of DSS for 2 or 3 weeks followed by water, then euthanized at 4 weeks. Disease activity index (DAI), histological damage, and myeloperoxidase assay (MPO), as index of neutrophil infiltration, were evaluated. Cytokines and µOR mRNAs were measured with RT-PCR, and nuclear factor-kB (NF-kB), the antiapoptotic factor Bcl-xL, and caspase 3 and 7 with Western blot. KEY RESULTS: DSS induced acute colitis with elevated DAI, tissue damage, apoptosis and increased MPO, cytokines, µOR mRNA, and NF-kB. DAMGO significantly reduced DAI, inflammatory indexes, cytokines, caspases, and NF-kB, and upregulated Bcl-xL, effects prevented by µOR antagonist. In DSS mice plus 4 weeks of water, DAI, NF-kB, and µOR were normal, whereas MPO, histological damage, and cytokines were still elevated; DAMGO did not reduce inflammation, and did not upregulate Bcl-xL. CONCLUSIONS & INFERENCES: µOR activation ameliorated the acute but not the delayed phase of DSS colitis by reducing cytokines, likely through activation of the antiapoptotic factor, Bcl-xL, and suppression of NF-kB, a potentiator of inflammation.
Asunto(s)
Colitis/metabolismo , Inflamación/metabolismo , Receptores Opioides mu/metabolismo , Animales , Colitis/inducido químicamente , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Ratones , Ratones Endogámicos C57BL , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inhibidoresRESUMEN
BACKGROUND AND PURPOSE: Nitric oxide (NO) and vasoactive intestinal peptide (VIP) are considered transmitters of non-adrenergic, non-cholinergic (NANC) relaxations in guinea-pig trachea, whereas the role of carbon monoxide (CO) is unknown. This study was designed to assess the participation of CO, and to investigate the localization of haem oxygenase-2 (HO-2), the CO-producing enzyme, in tracheal neurons. EXPERIMENTAL APPROACH: NANC responses to electrical field stimulation (EFS) at 3 and 10 Hz were evaluated in epithelium-free whole tracheal segments as intraluminal pressure changes. Drugs used were: L-nitroarginine methyl ester (L-NAME, 100 microM) to inhibit NO synthase (NOS), alpha-chymotrypsin (2 U ml(-1)) to inactivate VIP, zinc protoporphyrin-IX (ZnPP-IX, 10 microM) to inhibit HO-2, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), a soluble guanylyl cyclase inhibitor. For immunohistochemistry, tissues were exposed to antibodies to PGP 9.5, a general neuronal marker, HO-2 and NOS, and processed with an indirect immunofluorescence method. KEY RESULTS: alpha-Chymotrypsin did not affect NANC relaxations. ODQ inhibited NANC responses by about 60%, a value similar to that obtained by combining L-NAME and ZnPP-IX. The combination of ODQ, L-NAME and ZnPP-IX reduced the responses by 90%. Subpopulations of HO-2 positive neurons containing NOS were detected in tracheal sections. CONCLUSIONS AND IMPLICATIONS: In the guinea-pig trachea, NANC inhibitory responses at 3 and 10 Hz use NO and CO as main transmitters. Their participation is revealed following inhibition of NOS, HO-2 and soluble guanylyl cyclase. The involvement of CO as a relaxing transmitter paves the way for novel therapeutic approaches in the treatment of airway obstruction.
Asunto(s)
Monóxido de Carbono/fisiología , Músculo Liso/fisiología , Tráquea/fisiología , Animales , Estimulación Eléctrica , Cobayas , Hemo Oxigenasa (Desciclizante)/fisiología , Inmunohistoquímica , Técnicas In Vitro , Isoenzimas/fisiología , Masculino , Relajación Muscular , Óxido Nítrico/fisiología , Péptido Intestinal Vasoactivo/fisiologíaRESUMEN
Intestinal ischemia impairs gastrointestinal motility. The aims of this study were to investigate the effect of intestinal ischemia on gastrointestinal transit and on the expression of enteric transmitters in the rat, and whether the glutamate N-methyl-d-aspartate receptors influence these effects. Ischemia (1 h), induced by occluding the superior mesenteric artery, was followed by 0 or 24 h of reperfusion. Normal and sham-operated rats served as controls. Serosal blood flow was measured with laser Doppler flow meter. Gastrointestinal transit was measured as time of appearance of a marker in fecal pellets. Immunohistochemistry was used to evaluate the number of neurons immunoreactive for neuronal nitric oxide synthase (NOS) or vasoactive intestinal polypeptide and the density of substance P immunoreactive fibers in the myenteric plexus. The N-methyl-d-aspartate receptors antagonist, (+)-5-methyl-10,11-dihydro-5HT-[a,b] cyclohepten-5,10-imine (MK-801) (1 mg/kg i.v.) or the NOS inhibitor, N-nitro-l-arginine (10 mg/kg i.v.) was administered prior to ischemia. Serosal blood flow was decreased by 70% during ischemia, but it was not altered in sham-operated rats. Gastrointestinal transit was significantly prolonged in ischemic/reperfused rats compared with controls. There was a significant increase in the number of vasoactive intestinal polypeptide and neuronal nitric oxide synthase immunoreactive neurons, and a marked decrease of substance P immunoreactive fibers in ischemia followed by 24 h of reperfusion animals compared with controls. These alterations were not observed in ischemia without reperfusion. A significant delay of gastrointestinal transit and increase of vasoactive intestinal polypeptide neurons were also observed in sham-operated rats. The changes in transmitter expression and gastrointestinal transit in ischemic/reperfused rats were prevented by pre-treatment with the NOS inhibitor, N-nitro-l-arginine or the N-methyl-d-aspartate receptors antagonist, MK-801. This study suggests an involvement of the glutamatergic system and its interaction with nitric oxide in intestinal ischemia/reperfusion. Ischemia/reperfusion might induce local release of glutamate that activates N-methyl-d-aspartate receptors leading to increased production of nitric oxide and adaptive changes in enteric transmitters that might contribute to gastrointestinal dysmotility.
Asunto(s)
Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Tránsito Gastrointestinal/fisiología , Isquemia/fisiopatología , Plasticidad Neuronal/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/fisiología , Daño por Reperfusión/fisiopatología , Análisis de Varianza , Animales , Arginina/farmacología , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Motilidad Gastrointestinal/efectos de los fármacos , Motilidad Gastrointestinal/fisiología , Tránsito Gastrointestinal/efectos de los fármacos , Inmunohistoquímica/métodos , Isquemia/metabolismo , Flujometría por Láser-Doppler/métodos , Masculino , NG-Nitroarginina Metil Éster/farmacología , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Flujo Sanguíneo Regional/efectos de los fármacos , Flujo Sanguíneo Regional/fisiología , Daño por Reperfusión/metabolismo , Factores de Tiempo , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Exogenously administered galanin inhibits cholinergic transmission to the longitudinal muscle and reduces peristaltic efficiency in the guinea pig ileum with a mechanism partially mediated by galanin receptor 1 (GAL-R1). We investigated the effect of exogenous galanin 1-16, which has high affinity for GAL-R1, on the ascending excitatory reflex of the circular muscle elicited by radial distension in isolated segments of guinea pig ileum. We used a three-compartment bath that allows dissecting the ascending pathway into the oral (site of excitatory motor neurons), intermediate (site of ascending interneurons) and caudal compartment (site of intrinsic primary afferent neurons). Galanin 1-16 (0.3-3 micromol L(-1)) applied to the oral compartment inhibited in a concentration-dependent manner the ascending excitatory reflex elicited by the wall distension in the caudal compartment. This effect was antagonized by the GAL-R1 antagonist, RWJ-57408 (1 and 10 micromol L(-1)). By contrast, galanin 1-16 was ineffective when added to the intermediate or caudal compartment up to 3 micromol L(-1). GAL-R1 immunoreactive neurons did not contain neuron-specific nuclear protein, a marker for intrinsic primary afferent neurons. These findings indicate that GAL-R1s are present on motor neurons responsible for the ascending excitatory reflex, but not on ascending interneurons and intrinsic primary afferent neurons.
Asunto(s)
Íleon/inervación , Neuronas Motoras/metabolismo , Receptor de Galanina Tipo 1/metabolismo , Animales , Galanina/farmacología , Cobayas , Íleon/efectos de los fármacos , Inmunohistoquímica , Interneuronas/metabolismo , Masculino , Microscopía Confocal , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Plexo Mientérico/citología , Plexo Mientérico/metabolismo , Neuronas Aferentes/metabolismo , Técnicas de Cultivo de Órganos , Fragmentos de Péptidos/farmacología , Peristaltismo/fisiología , Reflejo/fisiologíaRESUMEN
Mu-, delta- and kappa-opioid receptors (ORs) mediate the effects of endogenous opioids and opiate drugs. Here we report (1) the distribution of muOR in the guinea-pig and human gastrointestinal tract in relation to endogenous ligands, to functionally distinct structures in the gut and to deltaOR and kappaOR; and (2) the ligand-induced muOR endocytosis in enteric neurones using in vitro and in vivo models. In the guinea pig, muOR immunoreactivity is confined mainly to the myenteric plexus. MuOR myenteric neurones are most numerous in the small intestine, followed by the stomach and the proximal colon. MuOR immunoreactive fibres are dense in the muscle layer and the deep muscular plexus, where they are in close association with interstitial cells of Cajal. This distribution closely matches the pattern of enkephalin. MuOR enteric neurones comprise functionally distinct populations of neurones of the ascending and descending pathways of the peristaltic reflex. In human gut, muOR immunoreactivity is localized to myenteric and submucosal neurones and to immune cells of the lamina propria. DeltaOR immunoreactivity is located in both plexuses where it is predominantly in varicose fibres in the plexuses, muscle and mucosa, whereas kappaOR immunoreactivity appears to be confined to the myenteric plexus and to bundles of fibres in the muscle. MuOR undergoes endocytosis in a concentration-dependent manner, in vitro and in vivo. Pronounced muOR endocytosis is observed in neurones from animals that underwent abdominal surgery that has been shown to induce delay in gastrointestinal transit. We can conclude that all three ORs are localized to the enteric nervous system with differences among species, and that muOR endocytosis can be utilized as a means to visualize enteric neurones activated by opioids and sites of opioid release.
Asunto(s)
Tracto Gastrointestinal/química , Narcóticos/análisis , Receptores Opioides/análisis , Adulto , Animales , Tracto Gastrointestinal/metabolismo , Cobayas , Humanos , Masculino , Persona de Mediana Edad , Narcóticos/metabolismo , Receptores Opioides/biosíntesisRESUMEN
Galanin actions are mediated by distinct galanin receptors (GAL-R), GAL-R1, -R2 and -R3. We investigated the role of GAL-R1 in gastric motility and the expression of GAL-R1 in the rat stomach. In vivo, in urethane-anaesthetized rats, galanin (equipotent for all GAL-Rs) induced a short inhibition of gastric motility, followed by increase in tonic and phasic gastric motility; the latter was significantly reduced by the GAL-R1 antagonist, RWJ-57408. Galanin 1-16 (high affinity for GAL-R1 and -R2) induced a long-lasting decrease of intragastric pressure, which was not modified by RWJ-57408. In vitro, galanin and galanin 1-16 induced increase of intragastric pressure that was not affected by RWJ-57408. Tetrodotoxin (TTX) did not suppress the galanin excitatory effect, whereas the effect of galanin 1-16 on gastric contraction was increased by TTX- or N-nitro-L-arginine, an inhibitor of nitric oxide synthase. GAL-R1 immunoreactivity was localized to cholinergic and tachykinergic neurons and to neurons immunoreactive for nitric oxide synthase or vasoactive intestinal polypeptide. This study suggests that an extrinsic GAL-R1 pathway mediates the galanin excitatory action, whereas an extrinsic, non GAL-R1 pathway is likely to mediate the galanin inhibitory effect in vivo. GAL-R1 intrinsic neurons do not appear to play a major role in the control of gastric motility.
Asunto(s)
Motilidad Gastrointestinal/fisiología , Receptor de Galanina Tipo 1/fisiología , Animales , Relación Dosis-Respuesta a Droga , Galanina/farmacología , Motilidad Gastrointestinal/efectos de los fármacos , Técnicas In Vitro , Masculino , Red Nerviosa/efectos de los fármacos , Red Nerviosa/fisiología , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Receptor de Galanina Tipo 1/agonistasRESUMEN
Galanin effects are mediated by distinct receptors, galanin receptor 1 (GAL-R1), GAL-R2 and GAL-R3. Here, we analyzed 1) the role of GAL-R1 in cholinergic transmission and peristalsis in the guinea-pig ileum using longitudinal muscle-myenteric plexus preparations and intact segments of the ileum in organ bath, and 2) the distribution of GAL-R1 immunoreactivity in the myenteric plexus with immunohistochemistry and confocal microscopy. Galanin inhibited electrically stimulated contractions of longitudinal muscle-myenteric plexus preparations with a biphasic curve. Desensitization with 1 microM galanin suppressed the high potency phase of the curve, whereas the GAL-R1 antagonist, RWJ-57408 (1 microM), inhibited the low potency phase. Galanin (3 microM) reduced the longitudinal muscle contraction and the peak pressure, and decreased the compliance of the circular muscle. All these effects were antagonized by RWJ-57408 (1 or 10 microM). RWJ-57408 (10 microM) per se did not affect peristalsis parameters in normal conditions, nor when peristalsis efficiency was reduced by partial nicotinic transmission blockade with hexamethonium. In the myenteric plexus, GAL-R1 immunoreactivity was localized to neurons and to fibers projecting within the plexus and to the muscle. GAL-R1 was expressed mostly by cholinergic neurons and by some neurons containing vasoactive intestinal polypeptide or nitric oxide synthase. This study indicates that galanin inhibits cholinergic transmission to the longitudinal muscle via two separate receptors; GAL-R1 mediates the low potency phase. The reduced peristalsis efficiency could be explained by inhibition of the cholinergic drive, whereas the decreased compliance is probably due to inhibition of descending neurons and/or to the activation of an excitatory muscular receptor. Endogenous galanin does not appear to affect neuronal pathways subserving peristalsis in physiologic conditions via GAL-R1.
Asunto(s)
Galanina/farmacología , Íleon/fisiología , Plexo Mientérico/efectos de los fármacos , Peristaltismo/fisiología , Receptor de Galanina Tipo 1/metabolismo , Acetilcolina/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Cobayas , Inmunohistoquímica , Microscopía Confocal , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Plexo Mientérico/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
Immunohistochemistry and confocal microscopy were used to investigate mu opioid receptor (muOR) internalization in enteric neurons of the guinea-pig ileum following abdominal surgery. The following surgical procedures were performed under halothane or isofluorane anesthesia: a) midline abdominal skin incision, b) laparotomy or c) laparotomy with intestinal manipulation. Gastrointestinal transit was evaluated by using a non-absorbable marker and measuring fecal pellet output. In neurons from normal and control (anesthesia alone) animals, muOR was predominantly at the cell surface. muOR endocytosis following skin incision was not significantly different from controls (21.2+/-3.5% vs. 13.7+/-2.1%, mean+/-S.E.M.), whereas it was significantly increased by laparotomy (46.5+/-6.1%; P<0.01 vs. controls) or laparotomy plus intestinal manipulation (40.5+/-6.1%; P<0.01 vs. controls) 30 min following surgery compared with controls. muOR endocytosis remained elevated at 4 h (38.6+/-1.2%; P<0.01 vs. controls), whereas it was similar to controls at 6 and 12 h (17.5+/-5.8% and 11.2+/-3.0%). muOR endocytosis occurred in cholinergic and nitrergic neurons. Gastrointestinal transit was significantly delayed by laparotomy or laparotomy plus intestinal manipulation (12.8+/-1.2 and 13.8+/-0.6 h vs. 7.0+/-0.5 in controls; P<0.01), but was not significantly changed by skin incision (8.2+/-0.6 h). The findings of the present study support the concept that the noxious stimulation caused by abdominal surgery induces release of endogenous opioids thus resulting in muOR endocytosis in neurochemically distinct enteric neurons. muOR internalization can serve as indirect evidence of opioid release and as a means to visualize neuronal pathways activated by opioids.
Asunto(s)
Endocitosis/fisiología , Sistema Nervioso Entérico/metabolismo , Íleon/metabolismo , Íleon/cirugía , Receptores Opioides mu/metabolismo , Abdomen/fisiología , Abdomen/cirugía , Animales , Sistema Nervioso Entérico/química , Cobayas , Masculino , Neuronas/química , Neuronas/metabolismo , Receptores Opioides mu/análisisRESUMEN
Immunohistochemistry and confocal microscopy were used to investigate endocytosis and recycling of the native mu opioid receptor (muOR) in enteric neurons. Isolated segments of the guinea-pig ileum were exposed to increasing concentrations of muOR agonists at 4 degrees C to allow ligand binding and warming to 37 degrees C for 0 min (baseline) to 6 h in ligand-free medium to allow receptor internalization and recycling. The endogenous ligand, [Met]enkephalin, and [D-Ala(2),MePhe(4),Gly-ol(5)] enkephalin (DAMGO), an opioid analog, and the alkaloids, etorphine and fentanyl, induced rapid internalization of muOR immunoreactivity in enteric neurons, whereas morphine did not. muOR internalization was prevented by muOR antagonists. Basal levels of muOR immunoreactivity in the cytoplasm were 10.52+/-2.05%. DAMGO (1 nM-100 microM) induced a concentration-dependent increase of muOR immunofluorescence density in the cytoplasm to a maximum of 84.37+/-2.26%. Translocation of muOR immunoreactivity in the cytoplasm was detected at 2 min, reached the maximum at 15-30 min, remained at similar levels for 2 h, began decreasing at 4 h, and was at baseline values at 6 h. A second exposure to DAMGO (100 nM) following recovery of internalized muOR immunoreactivity at the cell surface induced a translocation of muOR immunoreactivity in the cytoplasm comparable to the one observed following the first exposure (46.89+/-3.11% versus 43.31+/-3.80%). muOR internalization was prevented by hyperosmolar sucrose, phenylarsine oxide or potassium depletion, which inhibit clathrin-mediated endocytosis. muOR recycling was prevented by pre-treatment with bafilomycin A1, an acidotropic agent that inhibits endosomal acidification, but not by the protein synthesis inhibitor, cycloheximide. This study shows that native muOR in enteric neurons undergoes ligand-selective endocytosis, which is primarily clathrin-mediated, and recycles following endosomal acidification. Following recycling, muOR is activated and internalized by DAMGO indicating that recycled receptors are functional.
Asunto(s)
Analgésicos Opioides/farmacología , Endocitosis/efectos de los fármacos , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Íleon/efectos de los fármacos , Neuronas/efectos de los fármacos , Receptores Opioides mu/metabolismo , Somatostatina/análogos & derivados , Animales , Arsenicales/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Encefalinas/farmacología , Inhibidores Enzimáticos/farmacología , Cobayas , Íleon/metabolismo , Inmunohistoquímica , Ligandos , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Potasio/farmacología , Receptores Opioides mu/efectos de los fármacos , Somatostatina/farmacología , Sacarosa/farmacología , Factores de TiempoRESUMEN
The goal of this study was to engineer gastrin-producing G cells of the gastric antrum to produce insulin. A pGas-Ins chimeric gene in which the gastrin promoter drives expression of the human insulin gene was constructed and was validated by transient transfection of GH4 and AGS cells. RT-PCR analysis and sequencing revealed three forms of differentially spliced insulin mRNA in GH4 cells transiently transfected by pGas-Ins. Gas-Ins transgenic mice were generated utilizing this chimeric gene. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated expression of the human insulin gene specifically in antral G cells. Northern blot analysis demonstrated that the shortest of the insulin mRNA three forms is predominantly expressed in stomach tissue. RT-PCR analysis also showed expression of the transgene in colon, pancreas, and brain tissues that was undetectable by northern analysis. We conclude that gastrin promoter can be used for targeting expression of human insulin to antral G cells and that antral G cells can express human insulin. Further refining of the chimeric gene design is required to enhance expression.
Asunto(s)
Células Secretoras de Gastrina/metabolismo , Insulina/genética , Animales , Northern Blotting , Células Cultivadas , Quimera , Cartilla de ADN/química , Femenino , Gastrinas/biosíntesis , Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Antro Pilórico/citología , Antro Pilórico/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
G protein-coupled receptors are cell surface signal-transducing proteins, which elicit a variety of biological functions by the activation of different intracellular effector systems. Many of these receptors, including the mu-opioid receptor (mu OR), have been localized in the gastrointestinal tract. mu OR is the target of opioids and alkaloids, potent analgesic drugs with high potential for abuse. mu OR is expressed by enteric neurons, and it undergoes ligand-selective endocytosis. It is of clinical importance because it mediates tolerance and other major side effects of opiate analgesics, including impairment of gastrointestinal propulsion. An important observation of mu OR is its differential trafficking and desensitization properties in response to individual agonists, which might have long-term physiological consequences and be involved in the development of opiate side effects. Receptor activation by agonists is the basis for signaling, and alterations of the mechanisms controlling cellular responses of G protein-coupled receptors to agonists might be the basis of several diseases, including gastrointestinal diseases. Therefore, understanding these basic cellular mechanisms is important for developing appropriate therapeutic agents.
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Sistema Nervioso Entérico/metabolismo , Neuronas Motoras/metabolismo , Manejo del Dolor , Receptores Opioides mu/metabolismo , Animales , Endocitosis/fisiología , Humanos , Dolor/metabolismoRESUMEN
A monoclonal antibody (MAb) to galanin was prepared by cell fusion of myeloma Fox-NY and spleen cells from Robertsonian mice immunized with rat galanin. Hybridomas producing high-affinity antibodies were cloned in pristine-primed Balb/c mice. The antibody was purified by affinity chromatography and concentrated to 12 mg IgG/mL by dialysis. Immunoreactivity of the antibody was screened by radioimmunoassay. Ascites fluid contained approximately 10 mg/mL IgG that belong to the subclass of IgG2a as determined by enzyme-linked immunoadsorbent assay (ELISA). The titer of this IgG2a antibody entitled #G65G was 1:10,000 and the ID50 for rat galanin was 1000 fmol/mL as determined by liquid phase radioimmunoassay. Immunohistochemistry showed that this galanin MAb stains densed, beaded processes distributed to the enteric plexuses, where they appear to encircle neuronal cell bodies, to the muscle layer, where they are particularly abundant in the circular muscle layer and in the deep muscular layer, and to the mucosa. In vivo capacity of immunoneutralization by this antibody was tested in male Sprague-Dawley rats fasted for 24 h and anesthetized with urethane. Systemic injection of protein A purified galanin antibody (6 mg/rat) decreased by 70% of the inhibitory effect of intravenous galanin (2 nmol/kg/h i.v.) on gastric acid secretion induced by intracisternal TRH analog. These results show that galanin antibody #G65G is useful for in vivo immunoneutralization of galanin effects and is a valuable tool for immunohistochemical localization of galanin in gastrointestinal tissues.
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Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Galanina/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Galanina/administración & dosificación , Galanina/antagonistas & inhibidores , Ácido Gástrico/metabolismo , Hibridomas , Inmunoglobulina G , Isotipos de Inmunoglobulinas , Inmunohistoquímica , Intestinos/química , Masculino , Neuronas/química , Pruebas de Neutralización , Ratas , Ratas Sprague-DawleyRESUMEN
Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca(2+) mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.
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GTP Fosfohidrolasas/metabolismo , Receptores de Neuroquinina-1/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rab5/metabolismo , Animales , Calcio/metabolismo , Perros , Dinamina I , Dinaminas , Endocitosis , Técnica del Anticuerpo Fluorescente , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ratas , Transfección , Células Tumorales CultivadasRESUMEN
BACKGROUND & AIMS: In the intestine, tachykinins regulate motility by participating in neuromuscular and neuro-neuronal transmission. The aim of this study was to test the hypothesis that colonic propulsion is regulated by an interplay between tachykinergic and cholinergic transmission. METHODS: Propulsion was elicited by intraluminal distention of a thin rubber balloon, which traveled from the oral to the anal end of guinea pig isolated distal colon segments. The overall contribution of endogenous tachykinins to colonic propulsion was examined by blocking NK1, NK2, and NK3 receptors simultaneously. RESULTS: NK2-receptor blockade by MEN 11420 inhibited propulsion, whereas blockade of NK(1) by SR 140333 or of NK3 receptors by SR 142801 had minor effects on motility. Blockade of muscarinic or nicotinic receptors by hyoscine or hexamethonium decelerated peristalsis up to propulsion arrest. In the presence of partial muscarinic receptor blockade, the NK1-receptor antagonist SR 140333 and the NK2-receptor antagonist MEN 11420 markedly inhibited propulsion. Propulsion was also inhibited by the NK3-receptor antagonist SR 142801 in the presence of partial nicotinic receptor blockade. The simultaneous administration of the 3 tachykinin antagonists inhibited propulsion by 50%. CONCLUSIONS: This study demonstrates the existence of an interplay between tachykinergic and cholinergic pathways during peristalsis and the importance of endogenous tachykinins acting at multiple receptor sites in the control of colonic propulsion.
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Colon/fisiología , Peristaltismo/fisiología , Taquicininas/fisiología , Animales , Colon/efectos de los fármacos , Combinación de Medicamentos , Hexametonio/farmacología , Técnicas In Vitro , Masculino , Antagonistas Muscarínicos/farmacología , Antagonistas Nicotínicos/farmacología , Péptidos Cíclicos/farmacología , Peristaltismo/efectos de los fármacos , Piperidinas/farmacología , Quinuclidinas/farmacología , Escopolamina/farmacología , Taquicininas/antagonistas & inhibidoresRESUMEN
Imunocytochemical techniques were used to determine whether agonist-induced activation of mu-opioid receptors alters the number and distribution of mu-opioid receptor-positive cells in the rat cerebral cortex. In untreated rats, mu-opioid receptor immunoreactivity was localized to neuronal perikarya and dendrites and to neuropilar punctate structures. mu-Opioid receptor-positive neurons were mostly in layers II and III and exhibited a bipolar or bitufted morphology. In rats treated with the mu-opioid receptor agonist etorphine (0.1mg/kg intraperitoneally) and perfused after different survival periods, there was an enhancement of immunostaining for mu-opioid receptors observed at 15min, reaching a maximum at 60min, and which returned to normal at 480min. Etorphine-induced effects included an increase in the intensity of cellular and neuropil staining; statistical analysis showed that the number of mu-opioid receptor-positive cells in etorphine-treated groups was significantly higher than in controls or saline-treated rats. In animals that received both etorphine and the mu-opioid receptor antagonist naloxone, the pattern of mu-opioid receptors immunoreactivity was similar to that of untreated animals. This study shows that the number of mu-opioid receptor-positive cells is significantly increased following etorphine treatment and suggests that agonist treatment may be exploited to increased immunostaining of mu-opioid receptors and also of other G-protein coupled receptors.