RESUMEN
Corticotropin-releasing hormone (Crh), a 41-residue polypeptide, activates two G-protein-coupled receptors, Crhr1 and Crhr2, causing (among other transductional events) phosphorylation of the transcription factor Creb. The physiologic role of these receptors is only partially understood. Here we report that male, but not female, Crhr2-deficient mice exhibit enhanced anxious behaviour in several tests of anxiety in contrast to mice lacking Crhr1. The enhanced anxiety of Crhr2-deficient mice is not due to changes in hypothalamic-pituitary-adrenal (HPA) axis activity, but rather reflects impaired responses in specific brain regions involved in emotional and autonomic function, as monitored by a reduction of Creb phosphorylation in male, but not female, Crhr2-/- mice. We propose that Crhr2 predominantly mediates a central anxiolytic response, opposing the general anxiogenic effect of Crh mediated by Crhr1. Neither male nor female Crhr2-deficient mice show alterations of baseline feeding behaviour. Both respond with increased edema formation in response to thermal exposure, however, indicating that in contrast to its central role in anxiety, the peripheral role of Crhr2 in vascular permeability is independent of gender.
Asunto(s)
Ansiedad/genética , Eliminación de Gen , Receptores de Hormona Liberadora de Corticotropina/genética , Hormona Adrenocorticotrópica/sangre , Animales , Trastornos de Ansiedad/genética , Encéfalo/metabolismo , Corticosterona/sangre , Hormona Liberadora de Corticotropina/administración & dosificación , Hormona Liberadora de Corticotropina/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Edema/genética , Conducta Alimentaria/fisiología , Femenino , Calor/efectos adversos , Sistema Hipotálamo-Hipofisario/fisiología , Inyecciones Intraventriculares , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Fosforilación , Sistema Hipófiso-Suprarrenal/fisiología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Restricción Física , Factores Sexuales , Estrés Fisiológico/sangre , Estrés Fisiológico/metabolismo , Estrés Fisiológico/fisiopatología , Aumento de PesoRESUMEN
Corticotropin-releasing factor (CRF) and cholecystokinin (CCK), two highly colocalized neuropeptides, have been linked to the etiology of stress-related anxiety disorders. Recent evidence points to the possibility that some of the anxiogenic effects of the central CCK system take place through interplay with the CRF system. The aim of the present study was to examine the effects of chronic, mild activation of CRF receptor 1 (CRF(1)) on the central CCK system of the C57BL/6J mouse. As shown by in situ hybridization, real-Time PCR and immunohistochemistry, 5 days of intracerebroventricular (i.c.v.) injections of a subeffective dose (2.3 pmol) of cortagine, a CRF(1)-selective agonist, resulted in an increase in CCK mRNA levels and CCK(2) receptor immunoreactivity in several brain regions, such as amygdala and hippocampus, known to be involved in the regulation of anxiety. Mice with elevated endogenous central CCK tone exhibited significantly higher anxiety-like behaviors in the open-field task and elevated plus maze, and enhanced conditioned fear. These behavioral changes were reversed by i.c.v. administration of the CCK(2)-selective antagonist LY225910, after 5 days of priming with cortagine. Under the same conditions, the intraperitoneal administration of the CRF(1) antagonist antalarmin was ineffective. This result indicated that once the CCK system was sensitized by prior CRF(1) activation, it exhibited its anxiogenic effects, without influence by CRF(1), possibly because of its observed downregulation. In sum, our results provide a novel model for the interaction of the CRF and CCK systems contributing to the development of hypersensitive emotional circuitry.
Asunto(s)
Ansiedad/metabolismo , Colecistoquinina/metabolismo , Condicionamiento Clásico/fisiología , Conducta Exploratoria/fisiología , Receptores de Hormona Liberadora de Corticotropina/fisiología , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Colecistoquinina/genética , Condicionamiento Clásico/efectos de los fármacos , Hormona Liberadora de Corticotropina/administración & dosificación , Relación Dosis-Respuesta a Droga , Conducta Exploratoria/efectos de los fármacos , Miedo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Antagonistas de Hormonas/farmacología , Inyecciones Intraventriculares , Ratones , Ratones Endogámicos C57BL , Quinazolinonas/farmacología , ARN Mensajero/análisis , Receptor de Colecistoquinina B/antagonistas & inhibidores , Receptor de Colecistoquinina B/genética , Receptor de Colecistoquinina B/metabolismo , Receptores de Hormona Liberadora de Corticotropina/agonistas , Proteínas Recombinantes de Fusión/administración & dosificación , Estrés Fisiológico/fisiologíaRESUMEN
In previous work we have examined the nature of converting enzymes for proinsulin, proglucagon, and prosomatostatin-I (PSS-I) in secretory granules isolated from anglerfish islets. The purpose of the present study was to extend the examination of precursor conversion to islet microsomes and to compare prohormone processing, including that of PSS-I and prosomatostatin-II (PSS-II), in islet secretory granules and microsomes. Microsomes (rough endoplasmic reticulum [RER] and Golgi complex) and secretory granules were prepared from anglerfish islets by differential and discontinuous density-gradient centrifugation. Microsomes were further fractionated into Golgi- and RER-enriched subfractions. Lysed secretory granule or microsome preparations were incubated in the presence of a mixture of radioactively labeled islet prohormones. Extracts of products generated were subjected to analysis by gel filtration and high-pressure liquid chromatography. Accuracy of product cleavage was monitored by comparing high-pressure liquid chromatography retention times from the radiolabeled in vitro conversion products with the retention times of labeled products from tissue extracts. All converting activity in microsomes was found to be similar to that in granules in that it had a pH optimum near pH 5 and was inhibited by p-chloromercuribenzoate. No significant differences in the converting activity of Golgi complex- and RER-enriched subfractions of microsomes was observed. The proinsulin, proglucagon, and PSS-II converting-enzymes, which were found in islet secretory granules, were also present and membrane-associated in islet microsomes. However, converting activity for PSS-I was displayed only in secretory granules. This suggests that two or more separate enzymes are involved in processing PSS-I and PSS-II, and that these enzymes have either differential distribution or differential activity in RER/Golgi complex and secretory granules. The demonstration of converting enzyme activity in islet microsomes supports the proposal that these enzymes may be synthesized at the RER and are internalized along with the prohormones.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Endopeptidasas/metabolismo , Islotes Pancreáticos/metabolismo , Microsomas/metabolismo , Precursores de Proteínas/genética , Procesamiento Proteico-Postraduccional , Somatostatina/análogos & derivados , Animales , Fraccionamiento Celular , Gránulos Citoplasmáticos/ultraestructura , Peces , Islotes Pancreáticos/ultraestructura , Microsomas/ultraestructura , Somatostatina/genética , Tiamina Pirofosfatasa/metabolismoRESUMEN
Preprosomatostatin-I (PPSS-I) is processed in anglerfish islets to release a 14-residue somatostatin (SS-14). However, very little is known regarding other processing events that affect PPSS-I. This is the first study to identify and quantify the levels of nonsomatostatin products generated as a result of processing of this somatostatin precursor in living islet tissue. The products of PPSS-I processing in anglerfish islet tissue were identified in radiolabeling studies using a number of criteria. These criteria included immunoreactivity, specific radiolabeling by selected amino acids, radiolabel sequencing, and chromatographic comparison to isolated, structurally characterized fragments of anglerfish PPSS-I using reverse-phase high performance liquid chromatography. Intact prosomatostatin-I (aPSS-I) was isolated from tissue incubated with [3H]tryptophan and [14C]leucine. Significant 14C radioactivity was observed in the products of 11 of the first 44 sequencer cycles in positions consistent with the generation of a 96-residue prosomatostatin. These results indicate that signal cleavage occurs after the cysteine located 25 residues from the initiator Met of PPSS-I, resulting in a signal peptide 25 amino acids in length. Nonsomatostatin-containing fragments of the precursor were also found in tissue incubated with a mixture of 3H-amino acids. Only a small quantity of the dodecapeptide representing residues 69-80 in the prohormone was found (10 nmol/g tissue). Two other fragments of aPSS-I, also observed to be present in low abundance, were found to correspond to residues 1-27 (16 nmol/g tissue) and to residues 1-67 (7 nmol/g tissue) of aPSS-I. No evidence for the presence of the fragment corresponding to residues 29-67 was found. However, large quantities of SS-14 were observed (287 nmol/g tissue), indicating that the major site of aPSS-I cleavage is at the basic dipeptide immediately preceding SS-14. Recovery of much lower levels of the nonsomatostatin fragments of aPSS-I suggests that prohormone processing at the secondary sites identified in this study occurs at a low rate relative to release of SS-14 from aPSS-I.
Asunto(s)
Peces/metabolismo , Islotes Pancreáticos/metabolismo , Precursores de Proteínas/metabolismo , Somatostatina/biosíntesis , Somatostatina/metabolismo , Secuencia de Aminoácidos , Animales , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína/metabolismoRESUMEN
Human epidermoid carcinoma A431 cells in culture produce a soluble 105-kilodalton protein which, by the criteria of epidermal growth factor (EGF) binding, recognition by monoclonal and polyclonal antibodies to the EGF receptor, amino-terminal sequence analysis and carbohydrate content, is related to the cell surface domain of the EGF receptor. The high rate of production and the finding that with biosynthetic labeling the specific activity of this 105-kilodalton protein exceeds that of the intact receptor indicate that it is not derived from membrane-bound mature receptor but is separately produced by the cell. These cells thus separately synthesize an EGF receptor that is inserted into the membrane and an EGF receptor-related protein that is secreted.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Glicoproteínas/biosíntesis , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Carbohidratos/análisis , Línea Celular , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB , Glicoproteínas/análisis , Humanos , Cinética , Peso Molecular , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismoRESUMEN
Two rabbits immunized with a portion of glutamate receptor (GluR) subunit GluR3 (amino acids 245-457) exhibited seizure-like behaviors, suggesting that antibodies to GluR3 may modulate neuronal excitability. Using whole-cell recording, rabbit GluR3 antisera were tested on cultured fetal mouse cortical neurons. In a subset of kainate-responsive neurons, miniperfusion of antisera and IgG evoked currents that were blocked by CNQX. Immunoreactivity to synthetic peptides prepared to subregions GluR3A (amino acids 245-274) and GluR3B (amino acids 372-395) was present in both rabbit sera. Peptide GluR3B, but not GluR3A, specifically blocked antisera- and IgG-evoked currents. Similar receptor activation and anti-GluR3 reactivity was present in sera from patients with active Rasmussen's encephalitis, an intractable pediatric epilepsy. Thus, antibodies to GluR3 define a region involved in agonist binding and specific receptor activation. These data suggest that antibodies to neuronal receptors can function as agonists and that autoantibodies to GluRs may be highly specific neurotoxicants in some neurological diseases.
Asunto(s)
Anticuerpos/farmacología , Receptores de Glutamato/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Células Cultivadas , Conductividad Eléctrica , Embrión de Mamíferos , Humanos , Inmunoglobulina G/farmacología , Ácido Kaínico/farmacología , Riñón , Ratones , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Neuronas/fisiología , Fragmentos de Péptidos/inmunología , Receptores de Glutamato/química , Receptores de Glutamato/genética , Convulsiones/inducido químicamente , TransfecciónRESUMEN
Corticotropin-releasing factor (CRF) affects catecholamine production both centrally and peripherally. The aim of the present work was to examine the presence of CRF, its related peptides, and their receptors in the medulla of human and rat adrenals and their direct effect on catecholamine synthesis and secretion. CRF, urocortin I (UCN1), urocortin II (UCN2), and CRF receptor type 1 (CRF1) and 2 (CRF2) were present in human and rat adrenal medulla as well as the PC12 pheochromocytoma cells by immunocytochemistry, immunofluorescence, and RT-PCR. Exposure of dispersed human and rat adrenal chromaffin cells to CRF1 receptor agonists induced catecholamine secretion in a dose-dependent manner, an effect peaking at 30 min, whereas CRF2 receptor agonists suppressed catecholamine secretion. The respective effects were blocked by CRF1 and CRF2 antagonists. CRF peptides affected catecholamine secretion via changes of subplasmaliminal actin filament polymerization. CRF peptides also affected catecholamine synthesis. In rat chromaffin and PC12 cells, CRF1 and CRF2 agonists induced catecholamine synthesis via tyrosine hydroxylase. However, in human chromaffin cells, activation of CRF1 receptors induced tyrosine hydroxylase, whereas activation of CRF2 suppressed it. In conclusion, it appears that a complex intraadrenal CRF-UCN/CRF-receptor system exists in both human and rat adrenals controlling catecholamine secretion and synthesis.
Asunto(s)
Glándulas Suprarrenales/efectos de los fármacos , Catecolaminas/metabolismo , Hormona Liberadora de Corticotropina/farmacología , Receptores de Hormona Liberadora de Corticotropina/fisiología , Glándulas Suprarrenales/metabolismo , Animales , Catecolaminas/biosíntesis , Células Cultivadas , Células Cromafines/metabolismo , Femenino , Humanos , Células PC12 , Ratas , Ratas Sprague-Dawley , Receptores de Hormona Liberadora de Corticotropina/agonistas , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Distribución Tisular , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , UrocortinasRESUMEN
Synthetic ovine corticotropin-releasing factor (CRF) was administered to normal male volunteer subjects as an intravenous bolus or 30-s infusion. Doses of CRF ranging from 0.001 to 30 micrograms/kg body wt were administered, and plasma immunoreactive (IR)-ACTH and IR-cortisol concentrations were measured. The threshold dose appeared to be 0.01-0.03 micrograms/kg, the half-maximal dose 0.3-1 micrograms/kg, and the maximally effective dose 3-10 micrograms/kg. Basal concentrations of IR-ACTH and IR-cortisol were 14 +/- 7.6 pg/ml (mean +/- SD) and 5.6 +/- 2.2 micrograms/dl, respectively. IR-ACTH rose as early as 2 min after CRF injection, reached peak levels in 10-15 min, and declined slowly thereafter. IR-cortisol rose at 10 min or later and reached peak levels in 30-60 min. At a dose of 30 micrograms/kg, neither IR-ACTH nor IR-cortisol fell from peak levels of 82 +/- 21 pg/ml (mean +/- SE) and 23 +/- 1.4 micrograms/dl, respectively, during the 2-h course of the experiment, indicating that CRF has a sustained effect on ACTH release and/or a prolonged circulating plasma half-life. There was little or no increase in the levels of other anterior pituitary hormones. At doses of 1 microgram/kg and higher, facial flushing, tachycardia, and, in some subjects, a 15-29-mmHg decline in systemic arterial blood pressure were observed, even though blood volume was replaced and the subjects remained supine. These data indicate that synthetic ovine CRF is a very potent and specific ACTH secretagogue in man. Administered with caution until its vasomotor effects are more fully defined, CRF promises to be a safe and very useful investigative, diagnostic, and, possibly, therapeutic agent in man.
Asunto(s)
Hormona Adrenocorticotrópica/sangre , Hormona Liberadora de Corticotropina/farmacología , Hidrocortisona/sangre , Adulto , Animales , Presión Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Cara/irrigación sanguínea , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Radioinmunoensayo , Respiración/efectos de los fármacos , OvinosRESUMEN
One of the most remarkable features of the mammalian central nervous system is its ability to store large amounts of information for periods approaching a lifetime. However, during the aging process cognitive domains, such as long-term (declarative) memory and working memory decline in some, but by far not all individuals. It is essential to understand the physiological changes that cause memory decline and also to elucidate why preserved memory abilities vary so greatly across individuals and memory tasks. A generally accepted hypothesis has been that long-lasting activity-dependent changes in the efficacy of synaptic transmission in the mammalian brain are considered to be of fundamental importance for the storage of information. There is now a more detailed understanding of the changes in neuronal plasticity during aging at the molecular and systems levels. This review discusses recent findings on age-related changes in neuronal plasticity, which have opened up novel sites of action for therapeutic intervention.
Asunto(s)
Envejecimiento/fisiología , Trastornos de la Memoria/fisiopatología , Plasticidad Neuronal , Acetilcolina/metabolismo , Adenosina/metabolismo , Envejecimiento/metabolismo , Animales , Canales de Calcio Tipo L/metabolismo , Humanos , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Canales de Potasio/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The differential modulation of learning and anxiety by corticotropin-releasing factor (CRF) through CRF receptor subtypes 1 (CRFR1) and 2 (CRFR2) is demonstrated. As learning paradigm, context- and tone-dependent fear conditioning of the mouse was used. Injection of CRF into the dorsal hippocampus before training enhanced learning through CRFR1 as demonstrated by the finding that this effect was prevented by the local injection of the unselective CRFR antagonist astressin, but not by the CRFR2-specific antagonist antisauvagine-30 (anti-Svg-30). In contrast, injection of CRF into the lateral intermediate septum impaired learning through CRFR2, as demonstrated by the ability of antisauvagine-30 to block this effect. When antisauvagine-30 was injected alone into the lateral intermediate septum, learning was enhanced. Such tonic control of learning was not observed when astressin or antisauvagine-30 was injected into the dorsal hippocampus. Injection of CRF after the training into the dorsal hippocampus and the lateral intermediate septum also enhanced and impaired learning, respectively. Thus, it was indicated that CRF acted on memory consolidation. It was concluded that the observed effects reflected changes of associative learning and not arousal, attention, or motivation. Although a dose of 20 pmol human/rat CRF was sufficient to affect learning significantly, a fivefold higher dose was required to induce anxiety by injection into the septum. Immobilization for 1 hr generated a stress response that included the induction of anxiety through septal CRFR2 and the subsequent enhancement of learning through hippocampal CRFR1. The involvement of either receptor subtype was demonstrated by region-specific injections of astressin and antisauvagine-30.
Asunto(s)
Ansiedad/fisiopatología , Condicionamiento Psicológico/fisiología , Receptores de Hormona Liberadora de Corticotropina/fisiología , Estrés Psicológico/fisiopatología , Estimulación Acústica , Animales , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Química Encefálica/fisiología , Condicionamiento Psicológico/efectos de los fármacos , Hormona Liberadora de Corticotropina/farmacología , Relación Dosis-Respuesta a Droga , Miedo/fisiología , Hipocampo/química , Hipocampo/fisiología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos BALB C , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/farmacología , Receptores de Hormona Liberadora de Corticotropina/agonistas , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Núcleos Septales/química , Núcleos Septales/fisiologíaRESUMEN
The present study examined the involvement of the 5-HT(1A) receptors in classical fear conditioning using the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propyloamino)tetralin hydrobromide (8-OH-DPAT) and the selective "silent" 5-HT(1A) receptor antagonist (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclo- hexane carboxamide trihydrochloride (WAY 100635). The drugs were administered both subcutaneously and bilaterally into the dorsal hippocampus of male C57BL/6J mice. The training was performed in a single trial in which a tone was followed by a footshock. The retention of context- and tone-dependent fear was examined in separate tests conducted either 1 or 24 hr after training. Subcutaneous 8-OH-DPAT (0.1-1.0 mg/kg), when injected before but not after training, caused a dose-dependent impairment of contextual fear in both 1 and 24 hr tests, whereas tone-dependent fear was less affected. Pretraining intrahippocampal injections of 5.0 microg but not 1.0 microg 8-OH-DPAT caused a severe deficit in contextual fear when tested 24 hr after training. When injected both subcutaneously and intrahippocampally, 8-OH-DPAT induced the 5-HT syndrome, indicative of postsynaptic 5-HT(1A) receptor activation at the dose ranges that impaired fear conditioning. However, the behavioral changes induced by 8-OH-DPAT at the time of training could not account for inhibitory effects of 8-OH-DPAT on fear conditioning. Neither subcutaneous (0.03 mg/kg) nor intrahippocampal (0.5 microg per mouse) WAY 100635 altered context- or tone-dependent fear. However, subcutaneous WAY 100635 blocked both the 5-HT syndrome and the impairment of fear conditioning induced by subcutaneous or intrahippocampal 8-OH-DPAT. In contrast, intrahippocampal WAY 100635 blocked the impairment caused by intrahippocampal but not subcutaneous 8-OH-DPAT, indicating the involvement of extrahippocampal 5-HT(1A) receptors in fear conditioning. It is concluded that the deficits in fear conditioning induced by 8-OH-DPAT are a result of postsynaptic 5-HT(1A) receptor activation that interferes with learning processes operating at acquisition but not consolidation. Furthermore, the dorsohippocampal 5-HT(1A) receptors play an important but not exclusive role in the limbic circuitry subserving contextual fear conditioning.
Asunto(s)
Condicionamiento Clásico/fisiología , Miedo/fisiología , Receptores de Serotonina/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin/administración & dosificación , Estimulación Acústica , Análisis de Varianza , Animales , Conducta Animal/efectos de los fármacos , Condicionamiento Clásico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Vías de Administración de Medicamentos , Electrochoque , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Inyecciones Subcutáneas , Masculino , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Microinyecciones , Piperazinas/administración & dosificación , Piridinas/administración & dosificación , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina 5-HT1 , Retención en Psicología/efectos de los fármacos , Retención en Psicología/fisiología , Antagonistas de la Serotonina/administración & dosificación , Agonistas de Receptores de Serotonina/administración & dosificaciónRESUMEN
In a study of a possible substrate underlying morphine addiction, we examined NMDA receptor-mediated synaptic transmission of core nucleus accumbens neurons after chronic morphine treatment, using intracellular recording in a slice preparation of rat. We evoked pharmacologically isolated NMDA EPSCs by local stimulation and elicited inward currents by NMDA superfusion. In control slices, Mg(2+) and phorbol 12,13-diacetate (PDAc), a protein kinase C activator, strongly inhibited and increased, respectively, NMDA EPSC amplitudes. The PDAc effects were likely postsynaptic because PDAc enhanced the currents evoked by superfused NMDA to the same extent that it did the NMDA EPSCs. Chronic morphine treatment significantly decreased NMDA EPSC amplitudes and the sensitivity of NMDA EPSCs to Mg(2+) and PDAc, as well as the kinetics of the decay (inactivation rate) of the EPSCs (from 97 +/- 2.5 msec in untreated rats to 78.7 +/- 1.8 msec in slices from treated rats). One week after withdrawal, the Mg(2+) and PDAc effects were still significantly less than those in control slices. Interestingly, 1 week of withdrawal led to an increased NMDA EPSC inactivation rate compared with controls. These data demonstrate that chronic morphine treatment significantly alters NMDA receptor-mediated synaptic transmission in the accumbens, and these effects persist 1 week after withdrawal. These long-term effects may represent an important neuroadaptation in opiate dependence.
Asunto(s)
Morfina/farmacología , Narcóticos/farmacología , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Transmisión Sináptica/efectos de los fármacos , Vías Aferentes/efectos de los fármacos , Vías Aferentes/fisiología , Animales , Vías Eferentes/efectos de los fármacos , Vías Eferentes/fisiología , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Magnesio/farmacología , Masculino , Morfina/administración & dosificación , Núcleo Accumbens/metabolismo , Ésteres del Forbol/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Sinapsis/efectos de los fármacos , Sinapsis/fisiología , Factores de TiempoRESUMEN
The role of corticotropin-releasing factor in autonomic regulation of heart rate, heart rate variability and behavior responses was investigated in two genetic mouse models: corticotropin-releasing factor receptor 1-deficient mice, and corticotropin-releasing factor-transgenic mice overexpressing corticotropin-releasing factor. Heart rate was recorded by radio-telemetry during novelty exposure and auditory fear conditioning. Locomotor activity and freezing served as behavioral indices. Locomotor activity and heart rate were invariably increased in response to novelty exposure in both corticotropin-releasing factor receptor 1-deficient mice and littermate wild-type controls. The heart rate responses during retention of conditioned auditory fear and the exponential relationship between heart rate and heart rate variability were unaffected by genotype. Moreover, conditioned fear responses inferred from multiple behavioral measures including freezing did not differ between corticotropin-releasing factor receptor 1-deficient and corticotropin-releasing factor receptor 1 wild-type control mice. Corticotropin-releasing factor-transgenic mice exhibited markedly reduced locomotor activity during novelty exposure when compared with littermate wild-type controls. Baseline and novelty-driven heart rate was slightly elevated in corticotropin-releasing factor-transgenic mice, whereas the novelty-induced increase of heart rate was not different between genotypes. In contrast, corticotropin-releasing factor-transgenic mice did not display a heart rate response indicative of conditioned auditory fear. It is concluded that corticotropin-releasing factor receptor 1-deficiency does not affect heart rate adjustment and behavioral responses to acute fearful stimuli. The resiliency of behavioral and cardiovascular patterns elevation argues against the involvement of corticotropin-releasing factor receptor 1 in acute emotional regulation on these two functional levels despite an absent corticosterone elevation in corticotropin-releasing factor receptor 1-deficient mice. It is hypothesized that the lack of a conditioned heart rate response in corticotropin-releasing factor-transgenic mice is attributable to an impairment of cognitive function. The results are compared with those of corticotropin-releasing factor receptor 2-deficient mice, and the role of the corticotropin-releasing factor system in cardiovascular regulation is discussed.
Asunto(s)
Conducta Animal/fisiología , Frecuencia Cardíaca/fisiología , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Estrés Psicológico/fisiopatología , Animales , Condicionamiento Clásico/fisiología , Electrocardiografía , Miedo/fisiología , Ratones , Ratones Transgénicos , Actividad Motora/fisiologíaRESUMEN
Research into the biology of corticotropin-releasing factor (CRF) has been intensified significantly by the structural characterization of the CRF receptor (CRF-R). Two receptor subtypes, CRF-R1 and CRF-R2, and three functional splice variants of CRF-R2 have been discovered. It appears that ligand binding requires interaction of the N-terminal domain with one or two other extracellular domains of the CRF-R. In contrast to the mammalian CRF-R1, the frog CRF-R1 discriminates between naturally occurring CRF-like peptides.
RESUMEN
N-Methyl-D-aspartate (NMDA) receptor subunits were characterized with seven polyclonal antibodies. The antibodies were directed against NR1-A, NR2A-N1, and NR2C-N1, representing N-terminal sequences of the NR1, NR2A, and NR2C subunits, and against NR1-E, NR2A-C1, and NR2C-C1, derived from C-terminal sequences of these subunits. The anti-NR1-D antibody was raised against the putative internal loop of NR1. A size of 118 kDa was found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for NR1 (from rat brain) detected by anti-NR1-D and -NR1-E, but not anti-NR1-A. With the anti-NR1-A antibody, a 125-kDa protein was discovered that may represent a glutamate receptor not yet characterized. NR2A and NR2C were identified as proteins with sizes of 175 and 140 kDa, respectively. Enzymatic N-deglycosylation generated a 97-kDa protein from NR1, a 105-kDa protein from the 125-kDa protein, a 162-kDa protein from NR2A, and a 127-kDa protein from NR2C. In contrast to the deglycosylation product of the NR2A, the 97- and 127-kDa proteins derived from NR1 and NR2C, respectively, were found significantly smaller than the molecular masses of 103 and 141 kDa, respectively, predicted on the basis of DNA data. These products may represent truncated proteins. The tissue content of the NR1 and NR2A was high in bovine hippocampus and cortex but lower in the cerebellum. In contrast, NR2C was solely found in the cerebellum. The 125-kDa protein was highest in the cerebellum and cortex.
Asunto(s)
Química Encefálica , Fragmentos de Péptidos/inmunología , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/inmunología , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Especificidad de Anticuerpos , Baculoviridae/genética , Escherichia coli/genética , Datos de Secuencia Molecular , Neuroblastoma/química , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Empalme del ARN , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/clasificación , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Distribución Tisular , Transfección , Células Tumorales CultivadasRESUMEN
Rat corticotropin-releasing factor receptor 1 (rCRFR1) was produced either in transfected HEK 293 cells as a complex glycosylated protein or in the presence of the mannosidase I inhibitor kifunensine as a high mannose glycosylated protein. The altered glycosylation did not influence the biological function of rCRFR1 as demonstrated by competitive binding of rat urocortin (rUcn) or human/rat corticotropin-releasing factor (h/rCRF) and agonist-induced cAMP accumulation. The low production rate of the N-terminal domain of rCRFR1 (rCRFR1-NT) by transfected HEK 293 cells, was increased by a factor of 100 in the presence of kifunensine. The product, rCRFR1-NT-Kif, bound rUcn specifically (K(D) = 27 nM) and astressin (K(I) = 60 nM). This affinity was 10-fold lower than the affinity of full length rCRFR1. However, it was sufficiently high for rCRFR1-NT-Kif to serve as a model for the N-terminal domain of rCRFR1. With protein fragmentation, Edman degradation, and mass spectrometric analysis, evidence was found for the signal peptide cleavage site C-terminally to Thr(23) and three disulfide bridges between precursor residues 30 and 54, 44 and 87, and 68 and 102. Of all putative N-glycosylation sites in positions 32, 38, 45, 78, 90, and 98, all Asn residues except for Asn(32) were glycosylated to a significant extent. No O-glycosylation was observed.
Asunto(s)
Disulfuros/química , Receptores de Hormona Liberadora de Corticotropina/fisiología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Eliminación de Gen , Glicosilación , Humanos , Ligandos , Espectrometría de Masas , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína/fisiología , Ratas , Receptores de Hormona Liberadora de Corticotropina/química , Receptores de Hormona Liberadora de Corticotropina/genética , Receptores de Hormona Liberadora de Corticotropina/metabolismoRESUMEN
An enzyme that performs the conversion of anglerfish prosomatostatin-II (pro-SS-II) to anglerfish SS-28 has been identified using an improved two-dimensional electrophoresis procedure. The enzyme is a single chain 39 kDa polypeptide with its isoelectric point at pH 5.9. The converting enzyme has an acidic pH optimum, consistent with the lowered pH of the intracellular site of propeptide conversion. Secretory granule extracts were examined to determine the inhibitor sensitivity and pH optimum of the conversion of anglerfish pro-SS-II to anglerfish SS-28 in this organelle. Production of anglerfish SS-28 by secretory granules was maximal at pH 4.2 and was completely inhibited by the addition of pepstatin. Since pepstatin is a specific inhibitor of aspartyl proteases, these results indicate that the purified enzyme is a member of this enzyme family. This conclusion was supported by the data from partial amino acid sequence analysis. Because these results are consistent with the role of the purified enzyme in the in vivo production of anglerfish SS-28, the identified aspartyl protease has been termed the anglerfish SS-28-generating propeptide-converting enzyme.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Peces/metabolismo , Somatostatina/biosíntesis , Animales , Ácido Aspártico Endopeptidasas/química , Catepsina D/metabolismo , Cromatografía Líquida de Alta Presión , Gránulos Citoplasmáticos/enzimología , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Islotes Pancreáticos/enzimología , Focalización Isoeléctrica , Mapeo Peptídico , Precursores de Proteínas/biosíntesis , Somatostatina-28RESUMEN
A propeptide converting enzyme capable of producing somatostatin-14 has been identified and partially characterized from anglerfish pancreatic islets. Results from N-terminal protein sequence analysis indicate that this enzyme is a member of the kex2/furin/PC family. This observation provides strong corroborative evidence that the kex2/furin/PC protease family is involved in propeptide conversion. Comparison of the obtained protein sequence with the cDNA sequence of mammalian PC2 also suggests that the active enzyme is derived from a precursor by cleavage at a site containing four consecutive basic amino acids.
Asunto(s)
Islotes Pancreáticos/enzimología , Proproteína Convertasas , Proteínas de Saccharomyces cerevisiae , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismo , Somatostatina/biosíntesis , Secuencia de Aminoácidos , Animales , Peces , Furina , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Serina Endopeptidasas/clasificación , Serina Endopeptidasas/genética , Subtilisinas/metabolismoRESUMEN
Somatostatin was purified from anglerfish pancreatic islets using acetic acid extraction, gel filtration (Bio-Gel P-10), ion exchange chromatography (CM Bio-Gel A), and reversed phase high pressure liquid chromatography. The resulting peptide was characterized by RIA, bioassay, and determination of amino acid composition. Anglerfish islet somatostatin was found to possess an amino acid composition and immunological and biological activities equivalent to synthetic somatostatin. Sequence analyses revealed that the primary structure was H-Ala-Gly-cyclo-[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys]-OH. These results demonstrate that anglerfish islet somatostatin has the same primary structure as somatostatin from all other sources characterized to date.
Asunto(s)
Islotes Pancreáticos/análisis , Somatostatina , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Bioensayo , Peces , Radioinmunoensayo , Somatostatina/aislamiento & purificaciónRESUMEN
A 41-residue peptide purified as a corticotropin-releasing factor/beta-endorphin-releasing factor (CRF) in vitro was tested for its ability to stimulate the secretion of ACTH, beta-endorphin, and corticosterone in three animal groups: 1) unanesthesized rats bearing indwelling venous cannulae, 2) rats pretreated with chloropromazine plus morphine sulfate plus pentobarbital (CPZ-MS-Nb, and 3) rats with hypothalamic deafferentiations in the frontal and lateral retrochiasmatic areas. In all three bioassays iv administration of 0.1-10 micrograms CRF elicited a dose-related increase in plasma ACTH and beta-endorphin values over a 5- to 15-min period. Corticosterone secretion was also elevated but responded maximally with all doses of CRF tested. Pretreatment of CPZ-MS-Nb animals with 20 micrograms dexamethasone 4 h before assay abolished the CRF-induced hormone secretion. These data suggest that CRF may play a physiological role in the regulation of the hypothalamic-pituitary-adrenal axis.