RESUMEN
Recently three different neonatal extracorporeal membrane oxygenation (ECMO) circuits have been employed in our clinic. These circuits were compared for clotting and bleeding complications. Initially, we used an ECMO circuit containing a roller pump and venous bladder without severe complications. Manufacturing of circuit components was discontinued, necessitating the replacement of this circuit by a circuit with a centrifugal pump with 3/8 inch inlet and outlet. Acute increase of oxygenator resistance requiring emergency changeout became unexpectedly a regularly occurring complication. The increase in resistance was suspected to be caused by oxygenator clotting, although oxygenator function was preserved. To prevent this complication, we changed to a levitating centrifugal pump with 1/4 inch inlet and outlet, after which no oxygenator malfunction has been observed. Macroscopic and electron microscopic analysis demonstrates that small clots are formed within the circuit, presumably in or near the centrifugal pump, which are transported to the oxygenator and clog up the hollow fiber layer at the inlet side, barely penetrating the oxygenator beyond this first layer. Our results suggest that low blood velocities accompanied with recirculation of blood within or near the centrifugal pump and/or heat generation within the pump could contribute to the formation of these clots.
Asunto(s)
Oxigenación por Membrana Extracorpórea , Hemostáticos , Trombosis , Humanos , Recién Nacido , Coagulación Sanguínea , Oxigenación por Membrana Extracorpórea/efectos adversos , Oxigenación por Membrana Extracorpórea/métodos , Trombosis/etiología , Oxigenadores de Membrana/efectos adversosRESUMEN
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.
Asunto(s)
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
The motilin receptor (MTLR) is an important therapeutic target for the treatment of hypomotility disorders but desensitization may limit its clinical utility. The aim of this study was to investigate the role of the C-terminal tail of the MTLR in the desensitization, phosphorylation and internalization process. Three MTLR mutants, C-terminally truncated from amino acid 412 till 384 (MTLRDelta385), 374 (MTLRDelta375) or 368 (MTLRDelta369), were constructed and C-terminally tagged with an EGFP and stably expressed in CHO cells co-expressing the Ca(2+) indicator apoaequorin. Activity and desensitization were studied by measuring changes in motilin-induced luminescent Ca(2+) rises. Receptor phosphorylation was investigated by immunoprecipitation and MTLR-EGFP internalization was visualized by fluorescence microscopy. Truncation only reduced MTLR affinity and the efficacy to induce Ca(2+) luminescent responses of the MTLRDelta375-EGFP mutant. Furthermore, the region between amino acid 375 and 368 seems to be important for proper cell surface expression of the MTLR since receptors of the MTLRDelta369-EGFP mutant but not of the other mutants were found intracellularly in vesicles. Truncation of the receptor till amino acid 384 or 374 did neither affect desensitization nor internalization. In contrast phosphorylation of the MTLRDelta385-EGFP mutant was reduced by 80% but was not affected in the MTLRDelta375-EGFP mutant. In conclusion, MTLR desensitization and internalization is not dependent on the presence of the C-terminal tail. Truncation favors internalization via either phosphorylation-independent pathways or via phosphorylation of alternative sites in the receptor.
Asunto(s)
Calcio/metabolismo , Receptores de la Hormona Gastrointestinal/química , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de Neuropéptido/química , Receptores de Neuropéptido/metabolismo , Aequorina/metabolismo , Secuencia de Aminoácidos , Animales , Apoproteínas/metabolismo , Células CHO , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Fármacos Gastrointestinales/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Mediciones Luminiscentes , Microscopía Fluorescente , Datos de Secuencia Molecular , Motilina/farmacología , Mutación , Fosforilación , Pruebas de Precipitina , Estructura Terciaria de Proteína , Receptores de la Hormona Gastrointestinal/genética , Receptores de Neuropéptido/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ProteínaRESUMEN
The pharmacological properties of MA-2029, a novel motilin receptor antagonist, were investigated. In vitro, MA-2029 (1 to 30 nM) competitively inhibited motilin-induced contractions in isolated rabbit duodenal longitudinal muscle strips, with a pA2 value of 9.17+/-0.01 (n=5). However, contractile responses to acetylcholine and substance P were unaffected even at 1 microM of MA-2029. MA-2029 concentration-dependently inhibited the binding of [125 I]motilin to motilin receptors in a homogenate of rabbit colon smooth muscle tissue and membranes of HEK 293 cells expressing human motilin receptors. The pKi of MA-2029 was 8.58+/-0.04 in the rabbit colon homogenate (n=4) and 8.39 in the HEK 293 cells (mean of duplicate experiments). In vivo, orally-administered MA-2029 (3 to 30 mg/kg) dose-dependently inhibited colonic contractions induced by motilin (3 microg/kg, i.v.) in conscious rabbits. Inhibition was caused by all doses at 30 min after administration and by 10 mg/kg or more at 4 h after administration. The plasma concentration of MA-2029 correlated with its inhibitory effect. Furthermore, the oral administration of MA-2029 (0.3 to 3 mg/kg) also inhibited abdominal muscle contractions (an index of the visceral pain) induced by intravenous infusion of motilin (3 microg/kg/h) during colorectal distension in conscious rabbits. These results indicate that MA-2029 is an orally active, selective and competitive motilin receptor antagonist. It is suggested that this compound may be useful for gastrointestinal disorders associated with disturbed gastrointestinal motility such as irritable bowel syndrome.
Asunto(s)
Motilina/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Oligopéptidos/farmacología , Receptores de la Hormona Gastrointestinal/antagonistas & inhibidores , Receptores de Neuropéptido/antagonistas & inhibidores , Administración Oral , Animales , Unión Competitiva , Línea Celular , Colon/efectos de los fármacos , Colon/metabolismo , Relación Dosis-Respuesta a Droga , Duodeno/efectos de los fármacos , Duodeno/metabolismo , Motilidad Gastrointestinal/efectos de los fármacos , Humanos , Masculino , Motilina/metabolismo , Oligopéptidos/administración & dosificación , Oligopéptidos/farmacocinética , Dolor/tratamiento farmacológico , Dolor/fisiopatología , Dimensión del Dolor , ConejosRESUMEN
UNLABELLED: Studies with fragments of the gastrointestinal peptide, motilin, indicate that the C-terminal region of this peptide plays an important role in the desensitization of the motilin receptor (MTLR). AIM: To verify this hypothesis we studied the desensitization, phosphorylation and internalization induced by motilin analogues of different chain length with agonistic and antagonistic properties in CHO-MTLR cells. METHODS: We studied motilin [1-22], the [1-14] fragment, the analogues Phe(3)[1-22] and Phe(3)[1-14], and two putative antagonists, GM-109 and MA-2029 (modified 1-4 and 1-3 fragments). Activation and desensitization (2h preincubation with the motilin analogues 10muM) were studied in CHO-MTLR cells by an aequorin based luminescence assay. Phosphorylation was studied by immunoprecipitation and internalization was visualized in CHO-MTLR cells containing an enhanced green fluorescent protein (CHO-MTLR-EGFP). RESULTS: Motilin [1-22] and [1-14] were more potent than Phe(3)[1-22] and Phe(3)[1-14] (pEC(50): 9.77, 8.78, 7.36 and 6.65, respectively) to induce Ca(2+) release. GM-109 and MA-2029 were without agonist activity. [1-22] and Phe(3)[1-22] decreased the second response to motilin from 78+/-2% to 11+/-3% and 34+/-3% (P<0.001), respectively, whereas [1-14], Phe(3)[1-14], GM-109 and MA-2029 had no desensitizing effect (68+/-5%, 78+/-3%, 78+/-6% and 78+/-5%, respectively, P>0.05). The rank order of MTLR-phosphorylation was: [1-22]>[1-14]>Phe(3)[1-22]=Phe(3)[1-14]>GM-109=MA-2029. Only motilin [1-22] and [1-14] induced receptor MTLR-EGFP internalization as shown by a decrease in membrane fluorescence: 20+/-3% and 7+/-3%, respectively. CONCLUSION: The C-terminus of motilin enhances desensitization, phosphorylation and internalization of the MTLR while modifications of the N-terminus can favor a conformation of the receptor that is less susceptible to phosphorylation and internalization.
Asunto(s)
Endocitosis/efectos de los fármacos , Motilina/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de Neuropéptido/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Proteínas Fluorescentes Verdes/metabolismo , Ligandos , Fosforilación , Receptores de la Hormona Gastrointestinal/antagonistas & inhibidores , Receptores de Neuropéptido/antagonistas & inhibidoresRESUMEN
UNLABELLED: The motilin receptor (MTLR) represents a clinically useful pharmacological target, as agonists binding to the MTLR have gastroprokinetic properties. In order to compare the molecular basis for interaction of the MTLR with motilin and with the non-peptide motilin agonist, erythromycin-A (EM-A), the negatively charged E119 located in the third transmembrane (TM3) region was mutated to D (E119D) and Q (E119Q), respectively, and changes in activity of the mutant receptors were verified. METHODS: Each mutant receptor was stably transfected in CHO-cells containing the Ca2+ indicator apo-aequorin. Receptor activation in response to motilin, EM-A and their analogues was assessed by Ca2+-luminescense. RESULTS: In the E119Q mutant, the Ca2+ response to motilin and EM-A was abolished while in the E119D mutant it was reduced with 62% (motilin) and 81% (EM-A). The pEC50 values were shifted from 9.65+/-0.03 to 7.41+/-0.09 (motilin) and from 6.63+/-0.12 to 4.60+/-0.07 (EM-A). Acetylation of the N-terminal amine group as in [N-acetyl-Phe]1 mot (1-14), decreased the potency 6.3-fold (WT-MTLR) and 148-fold (E119D). Acetylation of EM-A enol ether induced a more pronounced shift in potency: 7943-fold (WT-MTLR) and 1413-fold (E119D). CONCLUSION: The comparable loss of affinity of the mutant receptors for motilin and EM-A indicate that these agonists both interact with the TM3 domain of the MTLR. The results with acetylated derivatives support an ionic interaction between E119 of the MTLR with the N+ of the desosamine sugar in EM-A, but not with the N+ of the free amine group in motilin.
Asunto(s)
Eritromicina/metabolismo , Motilina/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de Neuropéptido/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Células CHO , Cricetinae , Inmunohistoquímica , Datos de Secuencia Molecular , Receptores de la Hormona Gastrointestinal/química , Receptores de Neuropéptido/química , PorcinosRESUMEN
Motilin, a 22-amino acid gastrointestinal peptide, and ghrelin, the natural ligand of the growth hormone secretagogue receptor, form a new group of structurally related peptides. Several lines of evidence suggest that motilin and ghrelin are involved in the control of gastrointestinal motility by the activation of receptors on enteric neurons. The aim of this study was to look for the existence of motilin, ghrelin, and their respective receptors in the myenteric plexus of the guinea pig. We used longitudinal muscle/myenteric plexus (LMMP) preparations and cultures of myenteric neurons of the guinea pig ileum, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR). Most of the motilin-immunoreactive (IR; 72.8%) and motilin receptor-IR (68.9%) neurons were also positive for neuronal nitric oxide synthase (nNOS), 72.8% and 68.9%, few for choline acetyl transferase (ChAT), 11.4% and 11.9%, respectively. In contrast, ghrelin was mainly colocalized with ChAT (72.2%), and only 3.6% of ghrelin-positive cells showed nNOS-IR in the LMMP. Neither motilin nor the motilin receptor or ghrelin colocalized with calbindin. RT-PCR studies revealed motilin, ghrelin, and ghrelin receptor mRNA transcripts in LMMP preparations and in cultured myenteric neurons. In conclusion, this study, for the first time, provides direct evidence for the existence of motilin and ghrelin in myenteric neurons and suggests that both peptides may play a role in the activation of the enteric nervous system and hence in the regulation of gastrointestinal motility.
Asunto(s)
Motilina/metabolismo , Plexo Mientérico/metabolismo , Neuronas/metabolismo , Hormonas Peptídicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de Neuropéptido/metabolismo , Animales , Southern Blotting , Colina O-Acetiltransferasa/metabolismo , Femenino , Ghrelina , Cobayas , Inmunohistoquímica , Masculino , Motilina/genética , Óxido Nítrico Sintasa/metabolismo , Hormonas Peptídicas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de la Hormona Gastrointestinal/genética , Receptores de Ghrelina , Receptores de Neuropéptido/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R) with potent stimulatory effects on food intake. The aim of the present study was to investigate the effects of ghrelin on neuronal activity of hypothalamic glucose responding neurons. Single unit discharges in the lateral hypothalamic area (LHA), the ventromedial hypothalamic nucleus (VMH), and the parvocellular part of the paraventricular nucleus(pPVN) were recorded extracellularly by means of four-barrel glass micropipettes in anesthetized rats. The activity of glucose-sensitive neurons (GSNs) in the LHA, pPVN, and of glucoreceptor neurons (GRNs) in the VMH modulated by administration of ghrelin was analyzed. In the LHA, the majority of GSNs (17/25) increased in frequency due to ghrelin. Whereas the majority of VMH-GRNs (27/33) and pPVN-GSNs (9/13) was inhibited. The responses to ghrelin were abolished by pretreatment of [D-Lys-3]-GHRP-6, ghrelin receptor antagonist. These data indicate that the glucose responding neurons in the LHA, VMH, and pPVN are also involved in the orexigenic actions of ghrelin in the hypothalamic circuits, although AgRP/NPY neurons in the arcuate nucleus (ARC) are the primary targets of ghrelin.
Asunto(s)
Glucosa/farmacología , Hipotálamo/citología , Neuronas/efectos de los fármacos , Hormonas Peptídicas/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Recuento de Células/métodos , Distribución de Chi-Cuadrado , Interacciones Farmacológicas , Femenino , Ghrelina , Masculino , Neuronas/clasificación , Neuronas/metabolismo , Ratas , Ratas WistarRESUMEN
PURPOSE OF REVIEW: To summarize the recent findings. RECENT FINDINGS: Studies of changes in the plasma levels confirm the earlier concepts, but offer little proof of causal effect. It is increasingly realized that peptides produced in the gut have a paracrine role or an indirect effect via the gut-brain axis. Interest in prokinetic peptide agonists remains high despite the failure of two candidate drugs, but relamorelin and camicinal offer new hope. SUMMARY: We review the original studies published since January 2013 on peptides produced in the gut and with an effect on gastrointestinal motility.
Asunto(s)
Hormonas Gastrointestinales/metabolismo , Motilidad Gastrointestinal/fisiología , Tracto Gastrointestinal/fisiología , Hipotálamo/fisiología , Animales , Regulación del Apetito/fisiología , Modelos Animales de Enfermedad , Sistema Nervioso Entérico/fisiología , Hormonas Gastrointestinales/fisiología , Motilidad Gastrointestinal/efectos de los fármacos , Humanos , RatonesRESUMEN
1. Inflammation may affect subpopulations of neurons of the myenteric plexus. 2. In the present study the effect of trinitrobenzene sulphonic acid (TNBS) induced colitis on nitrergic, purinergic and adrenergic inhibitory neurotransmission was studied as well as the consequences of the related changes on the response of 5-HT agonists using these neurotransmitters to mediate their effect. 3. Strips from normal and colitis rabbits (135 mg kg(-1) TNBS) were subjected to electrical field stimulation (EFS, 0.3 ms, 6V, 0.5 - 32 Hz, 10 s train). The response was measured isometrically in the absence or presence of L-NAME, suramin, guanethidine, the 5-HT agonists (5-HT(1/5A/7): 5-carboxamidotryptamine (5-CT), 5-HT(2): alpha-methyl-5-HT, 5-HT(3): 2-methyl-5-HT, 5-HT(4): 5-methoxytryptamine (5-MeOT)) or a combination. 4. In normal strips L-NAME (1 - 32 Hz), suramin (0.5 - 2, 8 Hz) and guanethidine (4, 16, 32 Hz) increased the response to EFS. This effect was abolished in inflamed strips and was accompanied by a decrease in nNOS expression. 5. In normal strips all 5-HT agonists induced pronounced (5-CT, alpha-methyl-5-HT) or small (2-methyl-5-HT, 5-MeOT) inhibitory neural responses. In inflamed strips this was reversed to cholinergic excitatory responses. 6. The effect of inflammation on the 5-HT(4) response was mimicked by preincubation of normal strips with L-NAME or suramin. Accordingly, in inflamed strips L-NAME or suramin did not affect the excitatory effects of 5-MeOT. 7. TNBS-colitis abolishes nitrergic, purinergic and adrenergic neurotransmission. This reverses serotonergic inhibition into excitation.
Asunto(s)
Colitis/fisiopatología , Contracción Muscular , Inhibición Neural , Agonistas de Receptores de Serotonina/farmacología , Serotonina/fisiología , Ácido Trinitrobencenosulfónico , Animales , Colitis/inducido químicamente , Colitis/patología , Colon/efectos de los fármacos , Colon/inervación , Colon/patología , Colon/fisiopatología , Modelos Animales de Enfermedad , Estimulación Eléctrica/métodos , Inhibidores Enzimáticos/farmacología , Femenino , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Bloqueo Nervioso , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ConejosRESUMEN
Due to motilin's relation to the migrating motor complex (MMC), the physiology of motilin has been mostly studied in man and dog. The cat does not have an MMC pattern, and little is known about cat motilin. Therefore we identified the cat motilin precursor (GenBank accession no. AF127917) and developed a quantitative polymerase chain reaction (PCR) to explore its distribution in the gastrointestinal tract and in the central nervous system (CNS). The precursor is closely related to the dog precursor and consists of an open reading frame of 348bp encoding the signal peptide (25 amino acids), the motilin sequence (22 amino acids) and the motilin associated peptide (69 amino acids). One amino acid of the signal peptide was subject to gene polymorphism. Quantification of motilin messenger RNA (mRNA) was for the first time achieved. It is most abundant in the gastrointestinal tract, with the highest concentration in the duodenum, the lowest in the colon and is not detectable in the corpus. However an important expression was also observed in several regions of the CNS, except the striatum and cerebral cortex. The highest level was in the hypothalamus (although 23-fold lower than in the duodenum), the lowest level in the pons. Moderate levels were found in the thyroid. These data suggest that the physiological role of motilin may extend beyond its effect on gastrointestinal motility.
Asunto(s)
Perfilación de la Expresión Génica , Motilina/química , Motilina/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Química Encefálica , Gatos , Tracto Gastrointestinal , Humanos , Datos de Secuencia Molecular , Motilina/genética , Filogenia , Precursores de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
Treatment with the anti-inflammatory cytokine, interleukin-11 (IL-11), in rabbits with TNBS-colitis reduces tissue damage but does not normalize body weight loss despite an increase in plasma levels of motilin, known to stimulate food intake. We investigated whether IL-11 could increase plasma levels of the anorectic peptide, leptin in non-inflamed and inflamed rabbits. In addition, the effect of IL-11 and leptin on motilin mRNA expression in the T84 cell line was tested. Five days post-inflammation, weight loss amounted 10.7+/-1.2%, but plasma leptin and motilin levels were unaffected. During IL-11 treatment, weight loss remained and plasma leptin levels dose-dependently increased with 27+/-5% (4 microg/kg day) and 108+/-7% (720 microg/kg day). Motilin levels increased in parallel with 23+/-12% or 256+/-97%. In non-inflamed animals, a prompt decrease in weight (-11.9+/-1%) was observed after treatment with the highest dose of IL-11 and this was associated with an increase in plasma leptin (70+/-18%) and motilin levels (113+/-7%). Both IL-11 and leptin stimulated motilin mRNA expression in T84 cells with a different time profile. In conclusion, the increase in plasma leptin levels during IL-11 treatment induces wasting in normal rabbits and may be one of the major factors involved in the maintenance of body weight loss in rabbits with colitis. Increase of motilin expression by leptin may be part of a feedback mechanism.
Asunto(s)
Colitis/sangre , Interleucina-11/administración & dosificación , Leptina/sangre , Motilina/sangre , Pérdida de Peso/efectos de los fármacos , Animales , Línea Celular , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/sangre , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Motilina/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ConejosRESUMEN
Intestinal motilin is known to stimulate gastrointestinal motility. Recently, it was shown that the motilin gene and the motilin receptor are expressed in various regions of the brain. We studied whether motilin can activate pathways in the rat hippocampus to stimulate gastric motility. Gastric motility was monitored in conscious rats, whereas extracellular electrical activity recordings of the hippocampus were performed on anaesthetized rats to measure the influence of microinjection of motilin and CCK-8 into the hippocampus and into the cerebral ventricles. We found that neurons in the CA3 region of the hippocampus are sensitive to gastric distension, and that injection of motilin into the hippocampus increased the amplitude of gastric contractions by 35.3+/-6.8%, while CCK-8 injection inhibited motility by -27.3+/-6.8%. The hippocampal motilin-induced stimulation of gastric motility (30.6+/-5.5%) was completely abolished by subdiaphragmal vagotomy (-2.8+/-4.4%) but unaffected by the intravenously applied receptor blockers atropine, phentolamine and propranolol. In vivo extracellular recordings of gastric distension-responsive CA3 neurons revealed that intracerebroventricular administration of motilin increased firing while CCK-8 inhibited firing. These opposite effects of motilin and CCK-8 fit with the nature of the actions of these gut-brain peptides on gastric motility. Our findings suggest that the stimulation of gastric motility by motilin administered in the hippocampus reflects the existence of a functional interaction between the hippocampus and a vago-vagus reflex running via a noncholinergic and nonadrenergic efferent pathway.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Motilidad Gastrointestinal/efectos de los fármacos , Hipocampo/efectos de los fármacos , Motilina/administración & dosificación , Potenciales de Acción/fisiología , Animales , Femenino , Motilidad Gastrointestinal/fisiología , Hipocampo/fisiología , Masculino , Motilina/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ratas , Ratas WistarRESUMEN
AIM: Electrical stimulation of colonic muscles elicits a response during the stimulation period, and a transient excitation after the stimulus. Post-stimulus or "rebound" excitation has been linked to pathways involving inhibitory neurotransmitters, prostaglandins and substance P but the mechanism is incompletely understood. Because rabbit colitis is characterized by a loss of inhibitory neurotransmission we hypothesized it might affect the rebound response. Therefore we characterized rebound responses in non-inflamed and inflamed tissue by comparing the effect of antagonists/blockers of putative (nitric oxide [NO], ATP, substance P, prostaglandins) and new (serotonin) neurotransmitters. METHODS: Strips from rabbits with colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) were subjected to electrical field stimulation. Because rebound responses are more prominent under nonadrenergic noncholinergic (NANC) conditions, the effect of specific antagonists (N(omega)-nitro-L-arginine methyl ester (L-NAME), indomethacin, SR140333, methiothepin) on the rebound response was compared under normal and NANC conditions. RESULTS: NANC-conditions increased rebound responses in non-inflamed strips, but this effect was reduced or abolished in inflamed strips. Rebound responses were reduced by pretreatment with the NO-synthase inhibitor, L-NAME, under NANC conditions in non-inflamed strips but not affected in inflamed tissue. In contrast, the P(2) purine receptor antagonist, suramin, did not affect rebound responses in inflamed and non-inflamed strips. The effect of the cyclo-oxygenase inhibitor (COX), indomethacin, on rebound responses was reversed from excitatory to inhibitory by inflammation. Under NANC conditions rebound contractions were also reduced by the neurokinin-1 (NK(1)) antagonist, SR140333, both in normal and inflamed strips. The most pronounced reduction in rebound responses in inflamed and non-inflamed strips under normal conditions was observed with the 5-hydroxytryptamin (1,2) (5-HT(1,2)) antagonist, methiothepin. CONCLUSION: Rebound responses are mainly non-cholinergic and involve NO, substance P, serotonin and inhibitory prostaglandins. In inflamed tissue the nitrergic pathway is absent, excitatory prostaglandins prevail and the cholinergic and tachykinergic components are relatively more important. However there remains an important serotonergic contribution. Our data suggest that inflammation damages different neural pathways to a different extent and is most selective for nitrergic pathways.
Asunto(s)
Colitis/fisiopatología , Adenosina Trifosfato/fisiología , Animales , Colitis/inducido químicamente , Colitis/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Estimulación Eléctrica , Femenino , Técnicas In Vitro , Indometacina/farmacología , Masculino , Metiotepina/farmacología , Contracción Muscular , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiopatología , NG-Nitroarginina Metil Éster/metabolismo , Antagonistas del Receptor de Neuroquinina-1 , Óxido Nítrico Sintasa/antagonistas & inhibidores , Piperidinas/farmacología , Antagonistas del Receptor Purinérgico P2 , Quinuclidinas/farmacología , Conejos , Antagonistas de la Serotonina/farmacología , Sustancia P/fisiología , Suramina/farmacología , Ácido TrinitrobencenosulfónicoRESUMEN
Neuronal NO-synthase (nNOS) was investigated in rat longitudinal muscle/myenteric plexus (LM/MP) tissue at the cellular and subcellular level. Using preparations and double immune staining and light and electron microscopy, we concluded that, in these preparations, nNOS is only present in neuronal cells. However, in spite of numerous attempts to morphologically identify the NOS-containing subcellular structure, no firm conclusions were possible. Consequently, the problem was approached by biochemical methods including gradient centrifugation followed by analysis of the fractions. Using a protocol involving gentle homogenisation of the tissue, we found that about 10% of the nNOS immune reactivity was particle-bound confirming previous results (Biochem. Pharmacol. 60 (2000) 145). However, applying a different protocol including strong homogenisation, we now demonstrated that about 50% of the immune reactive nNOS was sedimentable. The results suggested that particulate nNOS is associated with one single subcellular structure, which is different from the plasma membrane, rough and smooth endoplasmic reticulum, mitochondria and lysosomes. The equilibrium sedimentation characteristics of the nNOS containing particles corresponded partly to those containing vasoactive intestinal polypeptide (VIP) or synaptobrevin. Application of non-equilibrium centrifugation conditions, however, demonstrated that almost no co-localisation occurred. We conclude that, in the LM/MP tissue, nNOS is about 50% particle-bound in a subcellular structure, which is different from the VIP-containing particle and from synaptobrevin-containing exocytotic particles.
Asunto(s)
Intestino Delgado/enzimología , Intestino Delgado/inervación , Músculo Liso/enzimología , Músculo Liso/inervación , Plexo Mientérico/enzimología , Óxido Nítrico Sintasa/metabolismo , Animales , Compartimento Celular/fisiología , Células Cultivadas , Inmunohistoquímica , Intestino Delgado/ultraestructura , Masculino , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Músculo Liso/ultraestructura , Plexo Mientérico/ultraestructura , Neuronas/enzimología , Neuronas/ultraestructura , Óxido Nítrico Sintasa de Tipo I , Orgánulos/enzimología , Orgánulos/ultraestructura , Proteínas R-SNARE , Ratas , Ratas Wistar , Fracciones Subcelulares/enzimología , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestructura , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
This study investigates the effect of theophylline along the rabbit gastrointestinal tract in comparison with the pharmacodynamic effect produced by the combined application of its three major metabolites. At concentrations up to 10(-3) m, theophylline relaxed, in a declining order from the lower oesophageal sphincter (LOS) to pylorus, all regions of the upper gastrointestinal tract, but only the ascending colon from the intestinal regions studied. At concentrations higher than 10(-3) m, instead of relaxing, theophylline strongly contracted the antrum and pylorus. In all three small intestinal regions, at concentrations up to 10(-3) m, theophylline produced a weak contraction, which at higher concentrations became very strong, and at 10(-2) m was comparable to that produced by a supramaximal dose of acetylcholine. The additive relaxing effect resulting from the combined application of the theophylline's metabolites was, from oesophagus to pylorus, weaker than that produced by theophylline, while on the ascending colon it was comparable to that of the parent drug. In contrast, the additive contractile effect of the metabolites on the three small intestinal regions was four to five times higher the one produced by theophylline. In conclusion, this study shows that the additive effect of the combined application of theophylline's major metabolites on the rabbit gastrointestinal tract plays a major role in the final response of the intestine, and a minor one in the final responses of the gastric regions, while both the parent drug and the metabolites contribute to the final responses of the oesophagus and LOS.
Asunto(s)
Broncodilatadores/farmacología , Tracto Gastrointestinal/efectos de los fármacos , Teofilina/farmacología , Acetilcolina/administración & dosificación , Acetilcolina/farmacología , Animales , Broncodilatadores/administración & dosificación , Broncodilatadores/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Tracto Gastrointestinal/metabolismo , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Conejos , Teofilina/administración & dosificación , Teofilina/metabolismoRESUMEN
The pharmacological properties of MA-2029, a selective and competitive motilin receptor antagonist, were investigated in conscious dogs after oral administration. Gastrointestinal contractile activity was recorded by chronically implanted force transducers. The proximal gastric volume was measured with a barostat under constant pressure. Gastric emptying was examined using the paracetamol absorption test. MA-2029 (0.3-10 mg/kg, p.o.) administered in the interdigestive state inhibited gastrointestinal contractions induced by motilin (3 microg/kg, i.v.) in a dose-dependent manner. MA-2029 (0.3-3 mg/kg, p.o.) also inhibited the occurrence of spontaneous phase III contractions, even though MA-2029 had no effect on basal gastrointestinal motility or basal gastric emptying even at 10 and 30 mg/kg p.o. The inhibitory effect of MA-2029 on motilin-induced gastrointestinal motility corresponded to its plasma concentration. Motilin (0.3 microg/kg/h, i.v. infusion) reduced the proximal gastric volume by about 50% of control during isobaric distension. This effect was also inhibited by MA-2029 (1-10 mg/kg, p.o.) in a dose-dependent manner. In the digestive state, injection of motilin (3 microg/kg, i.v.) induced diarrhea in 9 of 11 dogs. MA-2029 (1-30 mg/kg, p.o.) reduced the incidence of diarrhea induced by motilin in a dose-dependent manner. The results indicate that MA-2029 inhibits hypermotility induced by motilin in conscious dogs without having an effect on the basal gastrointestinal tone or gastric emptying rate. MA-2029 may be useful in treating gastrointestinal disorders in which the pathogenesis involves the elevation of circulating motilin.
Asunto(s)
Diarrea/prevención & control , Fundus Gástrico/efectos de los fármacos , Motilidad Gastrointestinal/efectos de los fármacos , Tono Muscular/efectos de los fármacos , Oligopéptidos/farmacología , Receptores de la Hormona Gastrointestinal/antagonistas & inhibidores , Receptores de Neuropéptido/antagonistas & inhibidores , Administración Oral , Animales , Diarrea/inducido químicamente , Perros , Relación Dosis-Respuesta a Droga , Femenino , Vaciamiento Gástrico/efectos de los fármacos , Fundus Gástrico/fisiología , Fármacos Gastrointestinales/administración & dosificación , Fármacos Gastrointestinales/farmacología , Motilidad Gastrointestinal/fisiología , Masculino , Motilina , Contracción Muscular/efectos de los fármacos , Tono Muscular/fisiología , Músculo Liso/efectos de los fármacos , Oligopéptidos/administración & dosificaciónRESUMEN
PURPOSE OF REVIEW: Motilin is a hormone produced from endocrine cells of the duodenal mucosa to help regulate motility of the digestive tract. This review discusses new findings on the potential impact of motilin in human medicine. RECENT FINDINGS: Motilin is a member of the peptide family that includes ghrelin whose cDNA also encodes a new candidate peptide, obestatin. Physiological interactions between these products will have to be explored. Pharmacological agents, agonists as well as antagonists, to motilin receptors are now emerging for clinical application. Motilin-receptor characterization, regarding its localization on nerves or muscles, as well as its biochemical mechanisms to sensitization for example, will be important steps in the design of future motilin agonists or antagonists. SUMMARY: Motilin is a fascinating hormone for the physiologist. Its interaction with the family member ghrelin and with obestatin will open new areas for basic research. Motilin-receptor agonists or antagonists could soon be part of the therapeutic arsenal of the clinician to improve digestive dysmotility.
Asunto(s)
Motilina/fisiología , Motilidad Gastrointestinal/efectos de los fármacos , Ghrelina/fisiología , Humanos , Contracción Muscular/fisiología , Músculo Liso/metabolismo , Neuronas/metabolismo , Péptidos/fisiología , Receptores de la Hormona Gastrointestinal/agonistas , Receptores de la Hormona Gastrointestinal/antagonistas & inhibidores , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de la Hormona Gastrointestinal/fisiología , Receptores de Neuropéptido/agonistas , Receptores de Neuropéptido/antagonistas & inhibidores , Receptores de Neuropéptido/metabolismo , Receptores de Neuropéptido/fisiologíaRESUMEN
BACKGROUNDS & AIMS: The motilin receptor (MTLR) is an important therapeutic target for treatment of hypomotility disorders. The negative outcome in clinical trials with the motilin agonist, ABT-229, indicated that desensitization may limit the therapeutic usefulness of motilides. We therefore compared the mechanisms involved in the intracellular trafficking of the MTLR after stimulation with motilin, erythromycin-A (EM-A) or ABT-229. METHODS: Desensitization was studied by measuring changes in Ca2+ rises and by receptor binding studies in CHO cells co-expressing the Ca2+ indicator apoaequorin and the MTLR, C-terminally tagged with EGFP. Receptor phosphorylation was studied by immunoprecipitation. MTLR-EGFP trafficking to organelles and translocation of beta-arrestins were visualized by fluorescence microscopy. RESULTS: Agonist-induced desensitization of the MTLR was due to receptor internalization with potencies (p-int50) in the order of: ABT-229 (8.3)>motilin (7.86)>EM-A (4.77) but with no differences in the internalization kinetics (t(1/2): approximately 25 min). The percentage cell surface receptor loss was more profound after exposure to ABT-229 (88+/-1%) than to motilin (63+/-10%) or EM-A (34+/-2%). For motilin and EM-A MTLR phosphorylation probably occurs via G protein-coupled receptor kinases while for ABT-229 phosphorylation was also protein kinase C dependent. All agonists translocated cytosolic beta-arrestin-2 with greater affinity to the plasma membrane than beta-arrestin-1. After internalization the MTLR co-localized with transferrin but not with cathepsin D. After stimulation with motilin and EM-A the t(1/2) for MTLR resensitization was 3h and 1h, respectively but amounted 26h for ABT-229. CONCLUSION: Our results suggest that the resensitization kinetics determine the desensitization properties of the motilin agonists.
Asunto(s)
Eritromicina/análogos & derivados , Eritromicina/farmacología , Motilina/farmacología , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de Neuropéptido/metabolismo , Animales , Arrestinas/genética , Arrestinas/metabolismo , Células CHO , Cricetinae , Cricetulus , Humanos , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transfección , beta-Arrestina 1 , Arrestina beta 2 , beta-ArrestinasRESUMEN
The present study, aimed to clarify whether the gastrointestinal adverse effects following administration of the bronchodilator theophylline are owing to the action of the drug itself or its metabolites, investigates the pharmacodymanic effects of theophylline's metabolites on the spontaneous contractility in the rabbit upper gastrointestinal tract. Comparative examination reveals that while two of the metabolites, namely 1-methylxanthine (1-MX) and 3-methylxanthine (3-MX), cause a similar, but less pronounced than the parent drug, concentration-dependent relaxation on the isolated oesophagus, lower oesophageal sphincter (LOS), fundus, antrum and pylorus, the remaining two metabolites, 1,3-dimethyluric acid (1,3-DMU) and 1-methyluric acid (1-MU), produce either a weak stimulating effect, or an even weaker relaxation. The relaxation which is muscle-mediated, non-adrenergic non-cholinergic (NANC) and nitric oxide (NO)-independent is probably mediated via inhibition of the metabolites on phosphodiesterases (PDEs), while a presynaptic cholinergic pathway is involved in the weak stimulating effect. The effects of all substances are additive. As a consequence, the net result of the cumulative action of all metabolites in the oesophagus, LOS, antrum and pylorus is, at 10(-3) m, comparable with that of theophylline, but in the fundus it is lower than that of the parent drug, because in the latter tissue the stimulating effect of 1,3-DMU and 1-MU counteracts the relaxing effect of the other two metabolites. However, combination of the parent drug with its metabolites leads to a considerable relaxation in all the gastrointestinal regions extending from the oesophagus to pylorus. Conclusively, upper gastrointestinal adverse effects following theophylline's administration are also because of theophylline's metabolites.