RESUMEN
Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in early and late developmental periods. In particular, the RNA-binding and cytoskeleton remodeling functions of FMRP may be differently modulated during development.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Factores de Edad , Análisis de Varianza , Animales , Animales Recién Nacidos , Encéfalo/patología , Células Cultivadas , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína Ácida Fibrilar de la Glía , Hipocampo/citología , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
The dopaminergic mesolimbic system plays a key role in mediating the reinforcing properties of ethanol and other drugs of abuse. Ethanol reinforcement and high alcohol drinking behaviour have been suggested to involve the ethanol-induced activation of endogenous opioid systems. Ethanol may alter opioidergic transmission at different levels, including opioid peptide biosynthesis and release, as well as binding to opioid receptors. The aim of this work was to investigate the effects of different ethanol doses on methionine-enkephalin (Met-enk) release from the rat nucleus accumbens (NAcc). Ethanol effects were also studied on Met-enk content in the NAcc, prefrontal cortex (PFC) and caudate-putamen (CP). Met-enk release was studied by microdialysis in Wistar anesthetized rats and peptide concentrations were quantitated by radioimmunoassay. Ethanol was administered by intraperitoneal injection after a 2-h basal release period. Ethanol doses of 0.5, 1 and 2.5 g/kg induced a 2.7-, 4.9- and 3.4-fold increase in Met-enk release from the NAcc. However, ethanol responses followed different kinetics, with earliest effects observed with the highest ethanol dose. In comparison, a 2.5-fold increase in peptide release was produced by 100 mM KCl. Ethanol, at a dose of 2.5 g/kg, induced a significant 66.7% decrease in Met-enk content in the NAcc, as well as a 76.4% reduction in peptide levels in the CP. Lower ethanol doses did not alter Met-enk content in these regions. On the other hand, an ethanol dose of 0.5 g/kg produced a non-significant decrease in Met-enk levels in the PFC. Our results suggest that ethanol-induced changes in enkephalin expression and release in regions of the mesocorticolimbic and nigrostriatal pathways could be involved in ethanol central effects. Released enkephalins by ethanol may modulate the dopaminergic activity of mesolimbic neurons and play a critical role in ethanol reinforcement mechanisms.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encefalina Metionina/metabolismo , Etanol/administración & dosificación , Neuronas/efectos de los fármacos , Análisis de Varianza , Animales , Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Inyecciones Intraperitoneales , Masculino , Microdiálisis , Neuronas/metabolismo , Radioinmunoensayo , Ratas , Ratas WistarAsunto(s)
Aborto Séptico/veterinaria , Proteínas Bacterianas/genética , Coxiella burnetii/genética , Enfermedades de las Cabras/microbiología , Polimorfismo Genético , Fiebre Q/veterinaria , Factores de Virulencia/genética , Aborto Séptico/microbiología , Animales , Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/inmunología , Biomarcadores , Coxiella burnetii/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Cabras , Reacción en Cadena de la Polimerasa , Embarazo , Fiebre Q/complicaciones , Fiebre Q/microbiología , Factores de Virulencia/inmunologíaRESUMEN
The preparation of a pure 125I-labeled monoiododerivative of mouse leptin is described. This radiolabeled analog has been used to characterize and localize central and peripheral leptin binding sites (Ob-R) of the mouse at different stages of its development. The affinity values found in membrane homogenates of various mouse tissues are similar and range between 0.1 and 0.3 nM, indicating that all the Ob-R isoforms have a similar affinity. Leptin binding sites are highly expressed at the membrane level in lung, intestine, kidney, liver, and skin and to a lesser degree in stomach, heart, and spleen. Brain, thymus, and pancreas homogenates are devoid of any specific binding. The distribution of mouse Ob-R has also been explored by autoradiography and dipping techniques on whole mouse sections. In lung, leptin binding sites are located at the pulmonary parenchyma and at the bronchiolar epithelial level. Binding sites are expressed all along the digestive tract from the tongue to the rectum (esophagus, stomach, intestine, colon, and rectum). In muscular visceral structures (stomach, intestine, and bladder) the binding is mainly present in the lamina propria. During development, leptin receptors are early expressed in the liver, kidney, and bone. In the lung, the Ob-R level increased gradually from birth to adulthood where the expression is maximal. By contrast, leptin receptors located in the medulla of the kidney remain remarkably constant all along the development. A broad signal is present in cartilage and bone particularly in vertebrae, limb, and ribs. Interestingly, leptin receptors are barely detectable in the mouse brain except in the choroid plexus and leptomeninges, whereas in the rat brain leptin binding sites are located in the thalamus, the piriform cortex, the cerebellum (at the granular and molecular cell layer), and the pineal gland.
Asunto(s)
Proteínas Portadoras/metabolismo , Leptina/metabolismo , Receptores de Superficie Celular , Animales , Animales Recién Nacidos , Autorradiografía , Sitios de Unión , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Cromatografía Líquida de Alta Presión , Embrión de Mamíferos , Humanos , Radioisótopos de Yodo , Ligandos , Pulmón/embriología , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , Obesidad/metabolismo , Especificidad de Órganos , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptores de Leptina , Proteínas Recombinantes/metabolismoRESUMEN
This study analyses the influence of the CCKergic system on the enkephalinergic system in the exploratory behavior of rats, using both behavioral and biochemical approaches. The results show that the increase of the spontaneous alternation behavior induced by the selective CCKB agonist, BC264 (3 microg/kg) was not suppressed by the opioid antagonists, naloxone (100 microg/kg), or naltrindole (300 microg/kg). In contrast, BC264 injected at the same dose induced a hyperlocomotor activity measured in the open-field test, which was antagonized by the selective delta opioid antagonist, naltrindole. BC264 (3 microg/kg) significantly increased the extracellular levels of Met-LI in the anterior part of the nucleus accumbens. Furthermore, local injection of naltrindole (0.25 microg/0.5 microl) in the anterior nucleus accumbens completely suppressed the hyperlocomotion induced by BC264. The behavioral effects induced by BC264 cannot be explained by its interaction with gastrinic receptors mediating gastric acid release, since BC264 produced a long-lasting increase of gastric acid output from conscious gastric fistula rats only at doses 100 times higher than those inducing behavioral modifications. The hyperlocomotion obtained after stimulation by BC264 of probably peripheral CCKB receptors, indicates that this receptor type could participate in the transmission of information between the peripheral system and some regions of the CNS involved in motivations and emotions.
Asunto(s)
Colecistoquinina/análogos & derivados , Núcleo Accumbens/metabolismo , Fragmentos de Péptidos/farmacología , Sistema Nervioso Periférico/efectos de los fármacos , Receptores de Colecistoquinina/metabolismo , Receptores Opioides delta/metabolismo , Animales , Colecistoquinina/farmacología , Conducta Exploratoria/efectos de los fármacos , Ácido Gástrico/metabolismo , Masculino , Microdiálisis , Naltrexona/análogos & derivados , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Núcleo Accumbens/efectos de los fármacos , Sistema Nervioso Periférico/metabolismo , Ratas , Ratas Wistar , Receptor de Colecistoquinina B , Receptores de Colecistoquinina/agonistas , Receptores Opioides delta/antagonistas & inhibidoresRESUMEN
Hemorphin generation by mice peritoneal macrophages has been recently reported, nevertheless no conclusive data exist to localize clearly the macrophage proteolytic activity implicated in their generation. Because lysosomes are believed to be the main site of degradation in the endocytic pathway, we have studied their potential implication in the generation of hemorphins from hemoglobin. When this protein is submitted to purified rat liver lysosomes, an early generation of hemorphin-7-related peptides, detected by a radioimmunoassay, was observed. These peptides seemed to be relatively stable during the first hours of hydrolysis.
Asunto(s)
Endopeptidasas/metabolismo , Hemoglobinas/biosíntesis , Hemoglobinas/metabolismo , Hígado/enzimología , Lisosomas/enzimología , Péptidos Opioides/biosíntesis , Fragmentos de Péptidos/biosíntesis , Animales , Hemoglobinas/aislamiento & purificación , Hígado/citología , Macrófagos/citología , Macrófagos/enzimología , Masculino , Péptidos Opioides/aislamiento & purificación , Fragmentos de Péptidos/aislamiento & purificación , Ratas , Ratas Wistar , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Using a specific rat monoclonal anti-idiotypic antibody we have examined the subcellular distribution of -opioid receptors in various neuronal subtypes of the rat spinal cord. The immunofluorescence was detected with a confocal microscope and in some cases serial images were processed for a three-dimensional (3-D) reconstruction of the neurons. Immunolabelling was found to be distributed throughout the spinal cord grey matter specially in the most superficial layers of the dorsal horn, around the central canal and in the region of motoneurons of the ventral horn. The 3-D reconstruction made on large neurons of lamina IX in the ventral horn and on neurons of lamina X around the central canal allowed the visualization of 5 -opioid receptors in the cytoplasm of the soma and proximal neurites of immunofluorescent neurons. Some immunolabelled receptors were also detected at the level of the plasma membrane of the cell bodies and in the nuclear matrix. Interestingly, a particular arrangement of delta-opioid receptors organized along parallel alignments was observed on the plasma membrane of some neurons. This study emphasizes the potential usefulness of a 3-D reconstruction in the study of the spatial arrangement of cellular components.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Neuronas/metabolismo , Neuronas/ultraestructura , Receptores Opioides delta/metabolismo , Médula Espinal/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Leucina Encefalina-2-Alanina/inmunología , Leucina Encefalina-2-Alanina/farmacocinética , Inmunohistoquímica , Microscopía Confocal , Neuronas Motoras/metabolismo , Neuronas Motoras/ultraestructura , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Médula Espinal/ultraestructura , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructuraRESUMEN
Mice transgenic with the human poliovirus receptor gene develop clinical signs and neuropathology similar to those of human poliomyelitis when neurovirulent polioviruses are inoculated into the central nervous system (CNS). Factors contributing to disease severity and the frequencies of paralysis and mortality include the poliovirus strain, dose, and gender of the mouse inoculated. The more neurovirulent the virus, as defined by monkey challenge results, the higher the rate of paralysis, mortality, and severity of disease. Also, the time to disease onset is shorter for more neurovirulent viruses. Male mice are more susceptible to polioviruses than females. TGM-PRG-3 mice have a 10-fold higher transgene copy number and produce 3-fold more receptor RNA and protein levels in the CNS than TGM-PRG-1 mice. CNS inoculations with type III polioviruses differing in relative neurovirulence show that these mouse lines are similar in disease frequency and severity, demonstrating that differences in receptor gene dosage and concomitant receptor abundance do not affect susceptibility to infection. However, there is a difference in the rate of accumulation of clinical signs. The time to onset of disease is shorter for TGM-PRG-3 than TGM-PRG-1 mice. Thus, receptor dosage affects the rate of appearance of poliomyelitis in these mice.
Asunto(s)
Proteínas de la Membrana , Poliomielitis/fisiopatología , Poliovirus/patogenicidad , Receptores Virales/metabolismo , Animales , Susceptibilidad a Enfermedades , Femenino , Humanos , Inyecciones Espinales , Masculino , Ratones , Ratones Transgénicos , Poliomielitis/virología , Poliovirus/genética , Receptores Virales/genética , Factores SexualesRESUMEN
Site-directed antibodies against synthetic related dermorphin peptides were previously produced and characterized. One of them, which specifically recognizes the crucial 'opioid message' (the N-terminal part of the dermorphin molecule (i.e. Tyr-D-Ala-Phe-Gly) was selected in order to detect and locate endogenous dermorphin-like molecules in rat, mouse and guinea pig tissues. Dermorphin-like peptides were found to be present in tissues known to contain peptides such as neurons in the central nervous system, nerve fibers in the gut and B and T immune cells. With all the tissues assayed, the HPLC profile obtained on the immunoreactive material showed the same main peak eluted at a retention time of 32 +/- 1 min. The results of biochemical experiments in which enzymatic treatments were performed on the dermorphin-like immunoreactivity indicate the immunoreactivity is a peptide resistant to aminopeptidase hydrolysis. This finding suggests the presence of a residue conferring resistance to proteolytic processes of this kind, which is likely to be a D-amino acid residue.
Asunto(s)
Aminopeptidasas/farmacología , Subgrupos Linfocitarios/química , Proteínas del Tejido Nervioso/análisis , Neuronas/química , Oligopéptidos/análisis , Animales , Química Encefálica , Cromatografía Líquida de Alta Presión , Colon/química , Colon/citología , Sistema Digestivo/química , Endopeptidasas/metabolismo , Cobayas , Técnicas para Inmunoenzimas , Masculino , Metionil Aminopeptidasas , Ratones , Ratones Endogámicos BALB C , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Neuroinmunomodulación , Neuropéptidos/análisis , Oligopéptidos/química , Oligopéptidos/metabolismo , Péptidos Opioides , Especificidad de Órganos , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Nervios Periféricos/química , Hipófisis/química , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Especificidad de la Especie , Bazo/químicaRESUMEN
Immunohistochemical investigations were carried out to determine the pattern of distribution of methionine- and leucine-enkephalin-like materials in the cat pylorus, duodenum, ileum and proximal and distal colon. The present results indicate that leucine-enkephalin-like materials are less densely distributed than methionine-enkephalin-like materials, but that the two patterns of distribution show some similarities. Considerable regional differences exist however in the distribution of these enkephalin-like materials in the muscular layers. In the duodenum, ileum and proximal colon, the immunoreactivity was mainly confined to the myenteric plexus and the circular muscle layer, where it was present in nerve cell bodies and in numerous fibres. In the longitudinal muscle and submucous layers, a few immunoreactive fibres were observed which sometimes surrounded blood vessels. In the pylorus and the distal colon, however, numerous immunoreactive fibres were observed in the longitudinal and circular muscle layers; the immunoreactivity was detected in the cell bodies of numerous myenteric plexus neurons but those of only a few submucous plexus neurons. In addition, the pylorus tissues contained immunoreactive plexi which were localized either within the longitudinal muscle or between the serosa and the longitudinal muscle layer. These plexi were connected to the myenteric plexus by immunoreactive nerve strands. In all the small intestinal segments studied, numerous immunoreactive varicosities were present in the deep muscular plexus, in the inner part of the circular muscle layer. Our results suggest that in cats, the nervous control of external muscular layers mediated by enkephalins shows regional differences. In the pylorus and the distal colon, it involves both the longitudinal and circular muscle layers, whereas in other intestinal segments, only the circular muscle layer is involved.
Asunto(s)
Sistema Digestivo/metabolismo , Encefalinas/metabolismo , Animales , Gatos , Colon/metabolismo , Duodeno/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Íleon/metabolismo , Masculino , Píloro/metabolismo , Distribución TisularRESUMEN
In order to investigate the putative physiological role of the in vivo release of hemorphins from hemoglobin in tissues, an immunological approach was developed. Specific and sensitive antiserum were raised against the C-part of the V-V-hemorphin-7. The antisera recognized to the same extent the related hemorphins V-V-hemorphin-7 and L-V-V-hemorphin-7. The validity of our immunological approach was analyzed by studying the in vitro release of hemorphin from hemoglobin by cathepsin D and compared to the pepsin hydrolysis. These two enzymes led to the release of these same products suggesting that cathepsin D acted as an accurate pepsin-like enzyme. Moreover, considering the poor sensitivity of the available methods of detection for the in vitro Cathepsin D activity, our specific and sensitive V-V-hemorphin-7 radioimmunoassay seems to be a useful alternative assay for this enzymatic activity.
Asunto(s)
Catepsina D/metabolismo , Hemoglobinas/metabolismo , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Bovinos , Cromatografía Líquida de Alta Presión , Reacciones Cruzadas , Hidrólisis , Sueros Inmunes , Pepsina A/metabolismo , Fragmentos de Péptidos/metabolismo , Radioinmunoensayo , Sensibilidad y EspecificidadRESUMEN
To detect and purify endogenous dermorphin-like molecules in mammalian tissues, an immunological approach was developed. Site-directed antibodies against synthetic dermorphin and related dermorphin peptides were produced. The immunogenic forms of dermorphin were selected to obtain antibodies recognizing different epitopes overlapping the whole dermorphin molecule. One of them specifically recognized the crucial "opioid message" (the N-terminal part of the molecule), which is required for a ligand to exert its full opioid activity. The validity of our immunological approach was analyzed by studying the dermorphin-related peptide distribution in Phyllomedusa sauvagei skin. The finding that tetrapeptide Y-A-G-F-OH was present in Phyllomedusa sauvagei extracts suggested that either the Tyr3-Pro6 peptidic bond may be relatively unstable or endogenous proteolytic enzymes present in Phyllomedusa skin may inactivate this peptidic bond.
Asunto(s)
Analgésicos Opioides/inmunología , Anuros , Epítopos , Oligopéptidos/inmunología , Piel/química , Analgésicos Opioides/metabolismo , Animales , Especificidad de Anticuerpos , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Motilidad Gastrointestinal/efectos de los fármacos , Cobayas , Íleon/efectos de los fármacos , Oligopéptidos/metabolismo , Péptidos Opioides , Radioinmunoensayo , Ensayo de Unión Radioligante , Ratas , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Análisis de SecuenciaRESUMEN
A polyclonal antiserum was produced against opioid binding sites using an anti-idiotypic approach whereby antibodies directed against the opioid agonist DSLET were used as immunogen. The anti-idiotypic antiserum recognized specific brain proteins with molecular masses of 76 +/- 4, 73 +/- 4 and 59 +/- 3 kDa, respectively. The immunolabeling of these proteins was mainly inhibited by mu, delta opioid agonists and a general antagonist, naloxone. The inhibition of immunoprecipitation by opioid agonists and antagonist and the developmental expression of these immunoreactive proteins found to occur during brain ontogeny strongly suggest that these three proteins were mu, delta but not kappa opioid binding sites. The anti-idiotypic antiserum both inhibits 3H-DADLE binding and mimics the inhibitory agonist effects on the stimulated cAMP level of NG 108-15 cells which expressed delta opiate receptors. Numerous mammalian brain opioid binding sites were labeled, due to the fact that the binding site was the epitope recognized by the anti-idiotypic antibodies. From the numerous studies performed with a view to characterizing the specificity of the anti-idiotypic antibodies, it was strongly suggested that the anti-idiotypic antibodies specifically recognize mu/delta opioid binding sites and they can therefore be powerful tools for studying the biochemical expression of these opioid binding sites in mammalian brains.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Química Encefálica , Receptores Opioides delta/análisis , Receptores Opioides mu/análisis , Animales , Sitios de Unión , AMP Cíclico/análisis , Encefalina Leucina/análogos & derivados , Encefalina Leucina/inmunología , Leucina Encefalina-2-Alanina/metabolismo , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Wistar , Receptores Opioides delta/inmunología , Receptores Opioides mu/inmunología , Células Tumorales CultivadasRESUMEN
The aim of the present study was to determine the distribution of methionine-enkephalin (ME) and leucine-enkephalin (LE) immunoreactivity in the sympathetic prevertebral ganglia (coeliac plexus and inferior mesenteric ganglion) and in the myenteric plexus-muscular layer complex of the digestive tract in guinea-pigs and rats. This study was performed using the same immunological approaches including radioimmunoassays and HPLC characterization as those used previously on cats in order to be able to make inter-region and inter-species comparisons. In rat and guinea-pig prevertebral ganglia, the distributions of the enkephalin immunoreactivities were comparable and were characterized by a low ME/LE concentration ratio, of less than 1. In the digestive tract of rats, the enkephalin immunoreactivities were homogeneously distributed, whereas in guinea-pigs, they were found to be very low in the lower oesophageal sphincter and high in the duodenum. In both species, the ME/LE concentration ratio was around 2. The ME/LE concentration ratio determined in the present study in peripheral nervous structures was much lower than that determined previously in the rat brain. Radioimmunoassay and biochemical data might indicate that different mechanisms are responsible for the processing and/or degradation of enkephalins in the central and peripheral nervous systems. The present study provides further evidences that there are tissue- and species-dependent differences in the distribution of enkephalin immunoreactivities. These differences should be taken into consideration when dealing with the effects and the role of enkephalins in the nervous control of intestinal motility in mammals.
Asunto(s)
Sistema Digestivo/metabolismo , Encefalinas/análisis , Ganglios Simpáticos/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Cobayas , Inmunoensayo , Ratas , Ratas WistarRESUMEN
Two opioid peptides were generated by in vitro pepsin treatment of bovine hemoglobin. These peptides were identified using a GPI test and purified using HPLC chromatographic techniques. They correspond to fragments 31-40 (LVV-hemorphin-7) and 32-40 (VV-hemorphin-7) of the beta-chain of bovine hemoglobin. Binding experiments strongly confirm that VV-hemorphin-7 and LVV-hemorphin-7 are opioid peptides since they inhibited [3H]naloxone binding to rat brain membranes. Our results indicate that VV-hemorphin-7 and LVV-hemorphin-7 exhibit a lesser potency both in GPI and binding tests. Selectivity and affinity of these purified peptides and synthetic hemorphin-7 for opioid receptors is discussed.
Asunto(s)
Hemoglobinas/metabolismo , Morfina/agonistas , Fragmentos de Péptidos/metabolismo , Receptores Opioides/agonistas , Animales , Unión Competitiva , Bovinos , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalina Leucina/análogos & derivados , Encefalina Leucina/farmacología , Encefalinas/farmacología , Cobayas , Hidrólisis , Íleon/metabolismo , Masculino , Naloxona/farmacología , Pepsina A/metabolismo , Ratas , Ratas Wistar , Sensibilidad y EspecificidadRESUMEN
Methionine-enkephalin-like immunoreactivity was detected in presynaptic nerve fibers and SIF cells in cat prevertebral ganglia. The immunoreactive nerve fibers contained a mixture of numerous small clear vesicles and a few large vesicles; the immunoreactivity was only confined to the large vesicles. Most of the immunoreactive fibers were in apposition with non-immunoreactive neuronal profiles, without any detectable synaptic membrane specializations. The other immunoreactive fibers formed synaptic contacts mainly with non-immunostained dendrites and to a lesser extent with axons and neuronal soma. The characterization at the ultrastructural level of the enkephalin-like immunoreactive structures is discussed as regards the modalities whereby opiates may be involved in sympathetic ganglionic transmission.
Asunto(s)
Gatos/anatomía & histología , Encefalina Metionina/análisis , Ganglios Simpáticos/química , Secuencia de Aminoácidos , Animales , Axones/química , Axones/ultraestructura , Gatos/metabolismo , Femenino , Ganglios Simpáticos/ultraestructura , Cobayas , Masculino , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Fibras Nerviosas/química , Fibras Nerviosas/ultraestructura , Especificidad de la Especie , Sinapsis/química , Sinapsis/ultraestructura , Transmisión SinápticaRESUMEN
The ultrastructural localization of delta-opioid receptors was studied using monoclonal anti-idiotypic antibody prepared with an anti-D-Ala2-D-Leu5-enkephalin. Immunocytochemical techniques were used on vibratome sections from rats perfused with paraformaldehyde. A high density of immunoreactivity was observed in the dorsal horn of the spinal cord, particularly the two superficial layers, the dorsolateral funiculus and the area surrounding the central canal. The labelling was absent when the antibody was preincubated with the immunogen. Competition between the anti-idiotypic antibody and different ligands, delta or mu, was controlled by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors for 3-4 h before addition of the anti-idiotypic antibody. Enkephalin, dermenkephalin and naltrindole induced disappearance of the labelling at 10(-9) M while dermorphin or dermorphin Lys7 were ineffective at the same concentration. Lamina II of the dorsal horn was studied by electron microscopy. The immunolabelling was mainly localized on cell membranes at appositions between the two neurons. About one third were localized between an axon terminal and a dendrite, the same proportion of labellings were between two axon terminals. Labelling was occasionally observed at appositions between a glomerular terminal and a dendrite or a terminal or at axoglial appositions. Axosomatic localizations were rare. The presynaptic localization of the labelling is in favor of a presynaptic mechanism of action for delta-opioids in the spinal cord, providing that these receptors are functional. delta-Opioid peptides probably act non-synaptically since receptors were never localized on synaptic differentiations.
Asunto(s)
Anticuerpos Antiidiotipos , Receptores Opioides delta/análisis , Médula Espinal/química , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Inmunohistoquímica , Microscopía Electrónica , Ratas , Ratas Wistar , Receptores Opioides delta/ultraestructuraRESUMEN
The opioïd system is implicated in mediating the effects produced upon administration of gamma-hydroxybutyrate. Gamma-hydroxybutyrate occurs endogenously in the mammalian brain, and is most probably involved in the regulation of some basic brain functions, particularly those concerning the dopaminergic nigrostriatal pathway, which is closely linked to the expression of enkephalins in the striatum. In the present study, in vivo microdialysis was used to examine the basic characteristics of methionine-enkephalin (met-enkephalin) release in the striatum of Wistar rats, using a high performance radioimmunoassay. Administration of gamma-hydroxybutyrate to the rats induced a dose-dependent decrease in the extracellular release of met-enkephalin. In parallel, a dose- and time-dependent gamma-hydroxybutyrate-induced accumulation of met-enkephalin in striatum was observed. These two phenomena (tissue accumulation and inhibition of release) were blocked by NCS-382, a gamma-hydroxybutyrate receptor antagonist. The striatal met-enkephalin accumulation does not seem to be exclusively due to the inhibition of its release. Thus, a gamma-hydroxybutyrate mediating effect on met-enkephalin synthesis is suggested, most probably occurring via functional modulation of striatal dopamine synthesis and release. To understand the role of this dopaminergic mechanism, unilateral lesions of the nigrostriatal dopaminergic pathway were carried out. In gamma-hydroxybutyrate-treated rats, striata exhibited a similar increase in met-enkephalin content. In untreated rats, only the lesioned striatum showed an identical increase in met-enkephalin levels. Thus, striatal met-enkephalin accumulation could be attributed to the suppression of the dopaminergic impulse flow, due to gamma-hydroxybutyrate or to the action of 6-hydroxydopamine. In the extracellular spaces (microdialysis experiments), gamma-hydroxybutyrate administration induced identical modifications of met-enkephalin release in lesioned or non-lesioned striata. These modifications could be reproduced by peripheral or striatal administration of sulpiride, a D2/D3 antagonist. From a functional point of view, the dopaminergic D2 receptor blockade or the gamma-hydroxybutyrate-induced inhibition of dopamine release could be considered to induce similar results, with identical consequences on striatal met-enkephalin accumulation and release. These results suggest that gamma-hydroxybutyrate-induced modifications in met-enkephalin release, presumably potentiated by 6-hydroxydopamine treatment, act via a functional modification of the nigrostriatal dopaminergic pathway.
Asunto(s)
Cuerpo Estriado/metabolismo , Encefalina Metionina/metabolismo , Oxibato de Sodio/farmacología , Ácido 3,4-Dihidroxifenilacético/análisis , Animales , Benzocicloheptenos/farmacología , Cuerpo Estriado/efectos de los fármacos , Dopamina/análisis , Masculino , Microdiálisis , Oxidopamina/toxicidad , Ratas , Ratas Wistar , Receptores Dopaminérgicos/efectos de los fármacos , Sulpirida/farmacología , Tetrodotoxina/farmacología , Veratridina/farmacologíaRESUMEN
In vitro pepsin treatment of plasma proteins generates biologically active peptides such as enkephalin-related peptides. These peptides were characterized using chromatographic techniques along with a radioimmunoassay procedure involving the use of Leu-enkephalin and Met-enkephalin antisera. Serum albumin is the only existing source of Met-enkephalin-immunoreactive peptides. One of these peptides consists of nine residues with the sequence NH2-Glu-Lys-Leu-Gly-Glu-Tyr-Gly-Phe-Gln; a second immunoreactive peptide might be the hexapeptide NH2-Gly-Glu-Tyr-Gly-Phe-Gln, which has been already identified in a rat serum albumin hydrolysate. Our results indicate that immunoglobulins constitute the main source of Leu-enkephalin-immunoreactive peptides. Immunoreactive NH2-Tyr-Phe-Leu was isolated from pepsin-treated bovine immunoglobulins. Binding experiments and cyclic nucleotide measurements suggested that this peptide was an enkephalin-related peptide. Similar experiments could be carried out to identify the proteins that contain enkephalin-like peptide sequences with the view to investigating the various biological processes occurring in enzymatically treated proteins.
Asunto(s)
Proteínas Sanguíneas/aislamiento & purificación , Encefalinas/aislamiento & purificación , Oligopéptidos/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/farmacología , Encéfalo/metabolismo , Bovinos , AMP Cíclico/metabolismo , Encefalinas/genética , Encefalinas/farmacología , Cobayas , Íleon/efectos de los fármacos , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Oligopéptidos/genética , Oligopéptidos/farmacología , Pepsina A , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptores Opioides/metabolismoRESUMEN
Cell bodies immunoreactive for methionine- and leucine-enkephalin are found in the area of the locus coeruleus (dorsolateral pons) of the cat after injection of colchicine in the ascending projections of the nucleus. Using radioimmunoassay procedures, it is shown that colchicine induces a significant increase in methionine- and leucine-enkephalin-immunoreactive material in this area of the brain. High pressure liquid chromatography analysis demonstrated that the immunoreactive materials were authentic methionine- and leucine-enkephalin. The methionine- and leucine-enkephalin patterns were identical in the colchicine injected and non-injected sides of the dorsolateral pons. It is suggested that, in this area of the brain, colchicine (i) does not significantly modify the processing of proenkephalin to form the pentapeptides methionine- and leucine-enkephalin, and (ii) does not induce the appearance of new substances reactive to the enkephalin antisera employed.