RESUMEN
Glioma refers to malignant central nervous system tumors that have histologic characteristics in common with glial cells. The most prevalent type, glioblastoma multiforme, is associated with a poor prognosis and few treatment options. On the basis of reports of aberrant expression of mGluR1 mRNA in glioma, evidence that melanoma growth is directly influenced by glutamate metabotropic receptor 1 (mGluR1), and characterization of ß-arrestin-dependent prosurvival signaling by this receptor, this study investigated the hypothesis that glioma cell lines aberrantly express mGluR1 and depend on mGluR1-mediated signaling to maintain viability and proliferation. Three glioma cell lines (Hs683, A172, and U87) were tested to confirm mGluR1 mRNA expression and the dependence of glioma cell viability on glutamate. Pharmacologic and genetic evidence is presented that suggests mGluR1 signaling specifically supports glioma proliferation and viability. For example, selective noncompetitive antagonists of mGluR1, CPCCOEt and JNJ16259685, decreased the viability of these cells in a dose-dependent manner, and glutamate metabotropic receptor 1 gene silencing significantly reduced glioma cell proliferation. Also, results of an anchorage-independent growth assay suggested that noncompetitive antagonism of mGluR1 may decrease the tumorigenic potential of Hs683 glioma cells. Finally, data are provided that support the hypothesis that a ß-arrestin-dependent signaling cascade may be involved in glutamate-stimulated viability in glioma cells and that ligand bias may exist at mGluR1 expressed in these cells. Taken together, the results strongly suggest that mGluR1 may act as a proto-oncogene in glioma and be a viable drug target in glioma treatment.
Asunto(s)
Glioma/genética , Glioma/patología , Receptores de Glutamato Metabotrópico/genética , Transducción de Señal/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cromonas/farmacología , Glioma/tratamiento farmacológico , Ácido Glutámico/genética , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Proto-Oncogenes Mas , Quinolinas/farmacología , ARN Mensajero/genética , Transducción de Señal/efectos de los fármacos , beta-Arrestinas/genéticaRESUMEN
The α4ß2 nicotinic acetylcholine receptor (nAChR) is important in central nervous system physiology and in mediating several of the pharmacological effects of nicotine on cognition, attention, and affective states. It is also the likely receptor that mediates nicotine addiction. This receptor assembles in two distinct stoichiometries: (α4)2(ß2)3 and (α4)3(ß2)2, which are referred to as high-sensitivity (HS) and low-sensitivity (LS) nAChRs, respectively, based on a difference in the potency of acetylcholine to activate them. The physiologic and pharmacological differences between these two receptor subtypes have been described in heterologous expression systems. However, the presence of each stoichiometry in native tissue currently remains unknown. In this study, different ratios of rat α4 and ß2 subunit cDNA were transfected into human embryonic kidney 293 cells to create a novel model system of HS and LS α4ß2 nAChRs expressed in a mammalian cell line. The HS and LS nAChRs were characterized through pharmacological and biochemical methods. Isolation of surface proteins revealed higher amounts of α4 or ß2 subunits in the LS or HS nAChR populations, respectively. In addition, sazetidine-A displayed different efficacies in activating these two receptor stoichiometries. Using this model system, a neurophysiological "two-concentration" acetylcholine or carbachol paradigm was developed and validated to determine α4/ß2 subunit stoichiometry. This paradigm was then used in layers I-IV of slices of the rat motor cortex to determine the percent contribution of HS and LS α4ß2 receptors in this brain region. We report that the majority of α4ß2 nAChRs in this brain region possess a stoichiometry of the (α4)3(ß2)2 LS subtype.
Asunto(s)
Corteza Motora/química , Receptores Nicotínicos/clasificación , Acetilcolina/farmacología , Animales , Células HEK293 , Humanos , Masculino , Subunidades de Proteína , Ratas , Ratas Sprague-Dawley , TransfecciónRESUMEN
The elemental anion chloride is generally considered a passive participant in neuronal excitability, and has never been shown to function as an agonist in its own right. We show that the antagonist-mediated, glutamate-independent inverse agonism of group II and III metabotropic glutamate (mGlu) receptors results from inhibition of chloride-mediated activation. In silico molecular modeling, site-directed mutagenesis, and functional assays demonstrate (1) that chloride is an agonist of mGlu3, mGlu4, mGlu6, and mGlu8 receptors with its own orthosteric site, and (2) that chloride is not an agonist of mGlu2 receptors. Molecular modeling-predicted and site-directed mutagenesis supported that this unique property of mGlu2 receptors results from a single divergent amino acid, highlighting a molecular switch for chloride insensitivity that is transduced through an arginine flip. Ultimately, these results suggest that activation of group II and III mGlu receptors is mediated not only by glutamate, but also by physiologically relevant concentrations of chloride.
Asunto(s)
Cloruros/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Secuencia de Aminoácidos , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Ácido Glutámico/farmacología , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutación Missense , Unión Proteica , Ratas , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismoRESUMEN
AT-1001 [N-(2-bromophenyl)-9-methyl-9-azabicyclo[3.3.1] nonan-3-amine] is a high-affinity and highly selective ligand at α3ß4 nicotinic cholinergic receptors (nAChRs) that was reported to decrease nicotine self-administration in rats. It was initially reported to be an antagonist at rat α3ß4 nAChRs heterologously expressed in HEK293 cells. Here we compared AT-1001 actions at rat and human α3ß4 and α4ß2 nAChRs similarly expressed in HEK 293 cells. We found that, as originally reported, AT-1001 is highly selective for α3ß4 receptors over α4ß2 receptors, but its binding selectivity is much greater at human than at rat receptors, because of a higher affinity at human than at rat α3ß4 nAChRs. Binding studies in human and rat brain and pineal gland confirmed the selectivity of AT-1001 for α3ß4 nAChRs and its higher affinity for human compared with rat receptors. In patch-clamp electrophysiology studies, AT-1001 was a potent partial agonist with 65-70% efficacy at both human and rat α3ß4 nAChRs. It was also a less potent and weaker (18%) partial agonist at α4ß2 nAChRs. Both α3ß4 and α4ß2 nAChRs are upregulated by exposure of cells to AT-1001 for 3 days. Similarly, AT-1001 desensitized both receptor subtypes in a concentration-dependent manner, but it was 10 and 30 times more potent to desensitize human α3ß4 receptors than rat α3ß4 and human α4ß2 receptors, respectively. After exposure to AT-1001, the time to recovery from desensitization was longest for the human α3ß4 nAChR and shortest for the human α4ß2 receptor, suggesting that recovery from desensitization is primarily related to the dissociation of the ligand from the receptor.
Asunto(s)
Agonismo Parcial de Drogas , Agonistas Nicotínicos/metabolismo , Oligopéptidos/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Femenino , Células HEK293 , Humanos , Masculino , Agonistas Nicotínicos/farmacología , Oligopéptidos/farmacología , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
Chronic nicotine administration increases the density of brain α4ß2* nicotinic acetylcholine receptors (nAChRs), which may contribute to nicotine addiction by exacerbating withdrawal symptoms associated with smoking cessation. Varenicline, a smoking cessation drug, also increases these receptors in rodent brain. The maintenance of this increase by varenicline as well as nicotine replacement may contribute to the high rate of relapse during the first year after smoking cessation. Recently, we found that sazetidine-A (saz-A), a potent partial agonist that desensitizes α4ß2* nAChRs, does not increase the density of these receptors in brain at doses that decrease nicotine self-administration, increase attention in rats, and produce anxiolytic effects in mice. Here, we investigated whether chronic saz-A and varenicline maintain the density of nAChRs after their up-regulation by nicotine. In addition, we examined the effects of these drugs on a measure of anxiety in mice and weight gain in rats. After increasing nAChRs in the rodent brain with chronic nicotine, replacing nicotine with chronic varenicline maintained the increased nAChR binding, as well as the α4ß2 subunit proteins measured by western blots. In contrast, replacing nicotine treatments with chronic saz-A resulted in the return of the density of nAChRs to the levels seen in saline controls. Nicotine, saz-A and varenicline each demonstrated anxiolytic effects in mice, but only saz-A and nicotine attenuated the gain of weight over a 6-week period in rats. These findings suggest that apart from its modest anxiolytic and weight control effects, saz-A, or drugs like it, may be useful in achieving long-term abstinence from smoking.
Asunto(s)
Ansiolíticos/uso terapéutico , Ansiedad/prevención & control , Azetidinas/uso terapéutico , Química Encefálica/efectos de los fármacos , Nicotina/toxicidad , Agonistas Nicotínicos/uso terapéutico , Piridinas/uso terapéutico , Receptores Nicotínicos/biosíntesis , Síndrome de Abstinencia a Sustancias/prevención & control , Tabaquismo/tratamiento farmacológico , Animales , Ansiolíticos/administración & dosificación , Ansiolíticos/farmacología , Ansiedad/inducido químicamente , Azetidinas/administración & dosificación , Azetidinas/farmacología , Benzazepinas/administración & dosificación , Benzazepinas/farmacología , Benzazepinas/uso terapéutico , Evaluación Preclínica de Medicamentos , Conducta Alimentaria/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Nicotina/administración & dosificación , Agonistas Nicotínicos/administración & dosificación , Agonistas Nicotínicos/farmacología , Piridinas/administración & dosificación , Piridinas/farmacología , Quinoxalinas/administración & dosificación , Quinoxalinas/farmacología , Quinoxalinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/genética , Cese del Uso de Tabaco , Tabaquismo/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Vareniclina , Aumento de Peso/efectos de los fármacosRESUMEN
Group II and group III metabotropic glutamate (mGlu) receptors are G protein-coupled receptors (GPCRs) that inhibit adenylyl cyclase via activation of Gαi/o. The purpose of this study was to design a universal method that overcomes previous challenges in consistently measuring group II and group III mGlu-receptor (mGluR) activation in stably transfected systems. In Chinese hamster ovary (CHO) cells stably transfected with the GloSensor cAMP biosensor, we optimized conditions for simple and highly reproducible (<5% S.E.M.) measurements of cAMP in real time. The GloSensor cAMP biosensor is a recombinant firefly luciferase conjugated to a cAMP-binding domain, where cAMP binding promotes a conformational shift within the GloSensor protein, inducing luciferase activity; cAMP levels are positively correlated with light output resulting from the luciferase-mediated breakdown of d-luciferin. Each group II and group III mGluR was then stably transfected into the CHO-GloSensor cell line, and experimental conditions were optimized for each receptor. During assay optimization, we observed ion sensitivity of several receptors and inverse agonist activity of the antagonist, LY341495 [2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-d-alanine]. Although these phenomena have been previously reported, they remain poorly understood, emphasizing the GloSensor assay as an important tool with which to study group II and group III mGlu receptors. Our results highlight many advantages of using the GloSensor method for measuring activation of group II and group III mGlu receptors, and they further suggest that corresponding methods designed to measure activation of any Gαi/o- or Gαs-coupled GPCR will be similarly advantageous.
Asunto(s)
Técnicas Biosensibles/métodos , AMP Cíclico/análisis , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Receptores de Glutamato Metabotrópico/fisiología , Aminoácidos/farmacología , Animales , Tampones (Química) , Células CHO , Técnicas de Cultivo de Célula , Membrana Celular/metabolismo , Colforsina/farmacología , Cricetinae , Cricetulus , AMP Cíclico/agonistas , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Ácido Glutámico/farmacología , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Ensayo de Unión Radioligante , Receptores de Glutamato Metabotrópico/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Transfección , Xantenos/farmacologíaRESUMEN
The metabotropic glutamate 1a (mGlu1a) receptor is a G protein-coupled receptor linked with phosphoinositide (PI) hydrolysis and with ß-arrestin-1-mediated sustained extracellular signal-regulated kinase (ERK) phosphorylation and cytoprotective signaling. Previously, we reported the existence of ligand bias at this receptor, inasmuch as glutamate induced both effects, whereas quisqualate induced only PI hydrolysis. In the current study, we showed that mGlu1 receptor agonists such as glutamate, aspartate, and l-cysteate were unbiased and activated both signaling pathways, whereas quisqualate and (S)-3,5-dihydroxyphenylglycine stimulated only PI hydrolysis. Competitive antagonists inhibited only PI hydrolysis and not the ß-arrestin-dependent pathway, whereas a noncompetitive mGlu1 receptor antagonist blocked both pathways. Mutational analysis of the ligand binding domain of the mGlu1a receptor revealed that Thr188 residues were essential for PI hydrolysis but not for protective signaling, whereas Arg323 and Lys409 residues were required for ß-arrestin-1-mediated sustained ERK phosphorylation and cytoprotective signaling but not for PI hydrolysis. Therefore, the mechanism of ligand bias appears to involve different modes of agonist interactions with the receptor ligand binding domain. Although some mGlu1a receptor agonists are biased toward PI hydrolysis, we identified two endogenous compounds, glutaric acid and succinic acid, as new mGlu1 receptor agonists that are fully biased toward ß-arrestin-mediated protective signaling. Pharmacological studies indicated that, in producing the two effects, glutamate interacted in two distinct ways with mGlu1 receptors, inasmuch as competitive mGlu1 receptor antagonists that blocked PI hydrolysis did not inhibit cytoprotective signaling. Quisqualate, which is biased toward PI hydrolysis, failed to inhibit glutamate-induced protection, and glutaric acid, which is biased toward protection, did not interfere with glutamate-induced PI hydrolysis. Taken together, these data indicate that ligand bias at mGlu1 receptors is attributable to different modes of receptor-glutamate interactions, which are differentially coupled to PI hydrolysis and ß-arrestin-mediated cytoprotective signaling, and they reveal the existence of new endogenous agonists acting at mGlu1 receptors.
Asunto(s)
Arrestinas/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Transducción de Señal/fisiología , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Ligandos , Receptores Acoplados a Proteínas G/fisiología , beta-ArrestinasRESUMEN
Nicotine-induced up-regulation of neuronal nicotinic receptors (nAChRs) has been known and studied for more than 25 years. Other nAChR ligands can also up-regulate nAChRs, but it is not known if these ligands induce up-regulation by mechanisms similar to that of nicotine. In this study, we compared up-regulation by three different nicotinic agonists and a competitive antagonist of several different nAChR subtypes expressed in HEK293 cells. Nicotine markedly increased α4ß2 nAChR binding site density and ß2 subunit protein. Carbachol, a known nAChR and muscarinic receptor agonist, up-regulated both α4ß2 nAChR binding sites and subunit protein 2-fold more than did nicotine. This increased up-regulation was shown pharmacologically to involve endogenously expressed muscarinic receptors, and stimulation of these muscarinic receptors also correlated with a 2-fold increase in α4 and ß2 mRNA. Muscarinic receptor activation in these cells appears to affect CMV promoter activity only minimally (â¼1.2 fold), suggesting that the increase in α4 and ß2 nAChR mRNA may not be dependent on enhanced transcription. Instead, other mechanisms may contribute to the increase in mRNA and a consequent increase in receptor subunits and binding site density. These studies demonstrate the possibility of augmenting nAChR expression in a cell model through mechanisms and targets other than the nAChR receptor itself.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Modelos Biológicos , Receptores Muscarínicos/biosíntesis , Receptores Nicotínicos/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores Muscarínicos/genética , Receptores Nicotínicos/genéticaRESUMEN
Chronic nicotine administration increases α4ß2 neuronal nicotinic acetylcholine receptor (nAChR) density in brain. This up-regulation probably contributes to the development and/or maintenance of nicotine dependence. nAChR up-regulation is believed to be triggered at the ligand binding site, so it is not surprising that other nicotinic ligands also up-regulate nAChRs in the brain. These other ligands include varenicline, which is currently used for smoking cessation therapy. Sazetidine-A (saz-A) is a newer nicotinic ligand that binds with high affinity and selectivity at α4ß2* nAChRs. In behavioral studies, saz-A decreases nicotine self-administration and increases performance on tasks of attention. We report here that, unlike nicotine and varenicline, chronic administration of saz-A at behaviorally active and even higher doses does not up-regulate nAChRs in rodent brains. We used a newly developed method involving radioligand binding to measure the concentrations and nAChR occupancy of saz-A, nicotine, and varenicline in brains from chronically treated rats. Our results indicate that saz-A reached concentrations in the brain that were â¼150 times its affinity for α4ß2* nAChRs and occupied at least 75% of nAChRs. Thus, chronic administration of saz-A did not up-regulate nAChRs despite it reaching brain concentrations that are known to bind and desensitize virtually all α4ß2* nAChRs in brain. These findings reinforce a model of nicotine addiction based on desensitization of up-regulated nAChRs and introduce a potential new strategy for smoking cessation therapy in which drugs such as saz-A can promote smoking cessation without maintaining nAChR up-regulation, thereby potentially increasing the rate of long-term abstinence from nicotine.
Asunto(s)
Azetidinas/farmacología , Conducta Animal/efectos de los fármacos , Química Encefálica/efectos de los fármacos , Piridinas/farmacología , Receptores Nicotínicos/efectos de los fármacos , Animales , Azetidinas/administración & dosificación , Azetidinas/sangre , Benzazepinas/administración & dosificación , Benzazepinas/sangre , Benzazepinas/farmacología , Sitios de Unión , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Membranas/efectos de los fármacos , Membranas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Nicotina/administración & dosificación , Nicotina/sangre , Nicotina/farmacología , Agonistas Nicotínicos/administración & dosificación , Agonistas Nicotínicos/sangre , Agonistas Nicotínicos/farmacología , Embarazo , Piridinas/administración & dosificación , Piridinas/sangre , Quinoxalinas/administración & dosificación , Quinoxalinas/sangre , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Autoadministración , Regulación hacia Arriba/efectos de los fármacos , VareniclinaRESUMEN
Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor that enhances the hydrolysis of membrane phosphoinositides. In addition to its role in synaptic transmission and plasticity, mGlu1 has been shown to be involved in neuroprotection and neurodegeneration. In this capacity, we have reported previously that in neuronal cells, mGlu1a exhibits the properties of a dependence receptor, inducing apoptosis in the absence of glutamate, while promoting neuronal survival in its presence (Pshenichkin, S., Dolinska, M., Klauzinska, M., Luchenko, V., Grajkowska, E., and Wroblewski, J. T. (2008) Neuropharmacology 55, 500-508). Here, using CHO cells expressing mGlu1a receptors, we show that the protective effect of glutamate does not rely on the classical mGlu1 signal transduction. Instead, mGlu1a protective signaling is mediated by a novel, G protein-independent, pathway which involves the activation of the MAPK pathway and a sustained phosphorylation of ERK, which is distinct from the G protein-mediated transient ERK phosphorylation. Moreover, the sustained phosphorylation of ERK and protective signaling through mGlu1a receptors require expression of beta-arrestin-1, suggesting a possible role for receptor internalization in this process. Our data reveal the existence of a novel, noncanonical signaling pathway associated with mGlu1a receptors, which mediates glutamate-induced protective signaling.
Asunto(s)
Arrestinas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transducción de Señal , Animales , Células CHO , Cricetinae , Cricetulus , Ácido Glutámico , Humanos , Fosforilación , Sustancias Protectoras , Ratas , Transfección , beta-Arrestina 1 , beta-ArrestinasRESUMEN
Nicotine increases the number of neuronal nicotinic acetylcholine receptors (nAChRs) in brain. This study investigated the effects of chronic nicotine treatment on nAChRs expressed in primary cultured neurons. In particular, we studied the chronic effects of nicotine exposure on the total density, surface expression and turnover rate of heteromeric nAChRs. The receptor density was measured by [¹²5I]epibatidine ([¹²5I]EB) binding. Untreated and nicotine-treated neurons were compared from several regions of embryonic (E19) rat brain. Twelve days of treatment with 10 µM nicotine produced a twofold up-regulation of nAChRs. Biotinylation and whole-cell binding studies indicated that up-regulation resulted from an increase in the number of cell surface receptors as well as intracellular receptors. nAChR subunit composition in cortical and hippocampal neurons was assessed by immunoprecipitation with subunit-selective antibodies. These neurons contain predominantly α4, ß2 and α5 subunits, but α2, α3, α6 and ß4 subunits were also detected. Chronic nicotine exposure yielded a twofold increase in the ß2-containing receptors and a smaller up-regulation in the α4-containing nAChRs. To explore the mechanisms of up-regulation we investigated the effects of nicotine on the receptor turnover rate. We found that the turnover rate of surface receptors was > 2 weeks and chronic nicotine exposure had no effect on this rate.
Asunto(s)
Neuronas/efectos de los fármacos , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/efectos de los fármacos , Animales , Biotinilación , Encéfalo/citología , Encéfalo/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Cicloheximida/farmacología , Femenino , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Inmunoprecipitación , Metionina/metabolismo , Embarazo , Inhibidores de la Síntesis de la Proteína/farmacología , Piridinas , Ratas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The objective of this study was to identify and quantify the heteromeric neuronal nicotinic receptors (nAChRs) in the rat hippocampus. The density of nAChR subtypes was assessed by labeling them with [(3)H]epibatidine ([(3)H]EB) followed by immunoprecipitation with subunit-selective antibodies. Sequential immunoprecipitation assays were used to establish associations between two different subunits, which then allowed the full subunit composition of the receptors to be deduced. Our results show that most of the hippocampal heteromeric nAChRs contain α4 and ß2 subunits. In fact, we identified two populations containing these two predominant subunits, the α4ß2 and α4ß2α5 subtypes which account for â¼ 40% and â¼ 35%, respectively, of the total [(3)H]EB-labeled receptors. An additional heteromeric subtype with the subunit composition of α4ß2α3 represented â¼ 10% of the total nAChRs, and another 10% of the immunoprecipitated receptors contained α4 and ß4 subunits, with or without the α3 subunit. To determine if α4ß2 and α4ß2α5 nAChR subtypes differ in their ligand binding affinities, the α3- and ß4-containing receptors were first removed by immunoprecipitation and then, competition studies with acetylcholine, nicotine, cytisine and sazetidine-A against [(3)H]EB were carried out on the remaining α4ß2 and α4ß2α5 subtypes. Results suggested these subtypes have comparable binding affinities for the nicotinic ligands used here.
Asunto(s)
Hipocampo/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Unión Competitiva/fisiología , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Inmunoprecipitación , Ligandos , Masculino , Agonistas Nicotínicos/metabolismo , Piridinas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
We used immunoprecipitation with subunit-specific antibodies to examine the distribution of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) that contain the alpha5 subunit in the adult rat brain. Among the regions of brain we surveyed, the alpha5 subunit is associated in approximately 37% of the nAChRs in the hippocampus, approximately 24% of the nAChRs in striatum, and 11-16% of the receptors in the cerebral cortex, thalamus, and superior colliculus. Sequential immunoprecipitation assays demonstrate that the alpha5 subunit is associated with alpha4beta2* nAChRs exclusively. Importantly, in contrast to alpha4beta2 nAChRs, which are increased by 37-85% after chronic administration of nicotine, the alpha4beta2alpha5 receptors are not increased by nicotine treatment. These data thus indicate that the alpha4beta2alpha5 nAChRs in rat brain are resistant to up-regulation by nicotine in vivo, which suggests an important regulatory role for the alpha5 subunit. To the extent that nicotine-induced up-regulation of alpha4beta2 nAChRs is involved in nicotine addiction, the resistance of the alpha4beta2alpha5 subtype to up-regulation may have important implications for nicotine addiction.
Asunto(s)
Encéfalo/efectos de los fármacos , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Subunidades de Proteína/metabolismo , Receptores Nicotínicos/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Animales , Sitios de Unión/efectos de los fármacos , Encéfalo/fisiología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Inmunoprecipitación/métodos , Piridinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/genéticaRESUMEN
The loss of more than half the number of GABA(A) receptors yet lack of pronounced phenotype in mice lacking the gene for the GABA(A) alpha1 subunit is somewhat paradoxical. We explored the role of tonic GABA(A) receptor-mediated current as a target of compensatory regulation in the alpha1 knock-out (-/-) mice. A 62% increase of tonic current was observed in the cerebellar granule cells (CGCs) of alpha1-/- compared with wild-type (+/+) mice along with a 67% increase of baseline current variance. Examination of whole-cell currents evoked by low concentrations of GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol suggested no upregulation of alpha6 and delta subunit-containing GABA(A) receptors in the alpha1-/-, confirming previous biochemical studies. Single-channel current openings were on average 32% shorter in the alpha1-/- neurons. Single-channel conductance and frequency of opening were not different between genotypes. Tonic current induced by application of the GABA transporter GAT-1 blocker NO711 (1-[2([(diphenylmethylene)imino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride) was significantly larger in the alpha1-/-, suggesting an increase of ambient GABA concentration. Experiments done with a known concentration of extracellular GABA complemented by a series of biochemical experiments revealed a reduction of GAT activity in alpha1-/- without an identifiable reduction of GAT-1 or GAT-3 protein. We report increased tonic GABA(A) receptor-mediated current in the alpha1-/- CGCs as a novel compensatory mechanism. Our data establish a role for GABA transporters as regulators of neuronal excitability in this and relevant models and examine other tonic conductance-regulating mechanisms responsible for the adaptive response of the cerebellar network to a deletion of a major synaptic GABA(A) receptor subunit.
Asunto(s)
Cerebelo/fisiología , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Potenciales de la Membrana/fisiología , Neuronas/fisiología , Receptores de GABA-A/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Células Cultivadas , Eliminación de Gen , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subunidades de Proteína , Receptores de GABA-A/química , Receptores de GABA-A/genética , Relación Estructura-ActividadRESUMEN
Responses to traumatic injury in the immature spinal cord may be different from those in adults. We modified an adult model of weight-drop injury to characterize the histopathology and functional recovery after spinal cord injury (SCI) in rat pups at postnatal day 14-15. A 10-g weight was dropped from 2.5 or 5.0 cm at T8-T9. Hindlimb function was evaluated at 24 h and 1, 2, 3, and 4 weeks after injury using the Combined Behavioral Score that estimates overall hind limb sensorimotor function, and the BBB scale for open field locomotion. Histopathology was examined at 15 min, 24 h, and 4 weeks after SCI. The initial hemorrhagic lesion was similar to that seen in adults, but the time course of secondary loss of ventral horn motor neurons was extended. By 4 weeks, only a partial rim of white matter surrounding a central cavity was seen. The 5.0 cm injury group exhibited significantly less recovery of function at 4 weeks than the 2.5 cm group. In the latter, the degree of hindlimb deficit at 4 weeks was similar to that previously described for adults with 10 g x 2.5 cm SCI. However, pups in both injury groups exhibited a significantly faster rate of recovery than adults. Recovery was maximal by 1 week after SCI in pups as compared to 3-4 weeks in adults. The more rapid functional recovery observed in the pups suggests that this new model may be useful for studying mechanisms of functional plasticity after SCI.
Asunto(s)
Envejecimiento/fisiología , Regeneración Nerviosa/fisiología , Plasticidad Neuronal/fisiología , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Factores de Edad , Animales , Modelos Animales de Enfermedad , Femenino , Miembro Posterior/inervación , Miembro Posterior/fisiopatología , Masculino , Trastornos del Movimiento/etiología , Trastornos del Movimiento/patología , Trastornos del Movimiento/fisiopatología , Degeneración Nerviosa/etiología , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Fibras Nerviosas Mielínicas/patología , Vías Nerviosas/lesiones , Vías Nerviosas/patología , Vías Nerviosas/fisiopatología , Parálisis/etiología , Parálisis/patología , Parálisis/fisiopatología , Ratas , Ratas Sprague-Dawley , Médula Espinal/patología , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/patología , Factores de TiempoRESUMEN
The majority of existing research on the function of metabotropic glutamate (mGlu) receptor 1 focuses on G protein-mediated outcomes. However, similar to other G protein-coupled receptors (GPCR), it is becoming apparent that mGlu1 receptor signaling is multi-dimensional and does not always involve G protein activation. Previously, in transfected CHO cells, we showed that mGlu1 receptors activate a G protein-independent, ß-arrestin-dependent signal transduction mechanism and that some mGlu1 receptor ligands were incapable of stimulating this response. Here we set out to investigate the physiological relevance of these findings in a native system using primary cultures of cerebellar granule cells. We tested the ability of a panel of compounds to stimulate two mGlu1 receptor-mediated outcomes: (1) protection from decreased cell viability after withdrawal of trophic support and (2) G protein-mediated phosphoinositide (PI) hydrolysis. We report that the commonly used mGlu1 receptor ligands quisqualate, DHPG, and ACPD are completely biased towards PI hydrolysis and do not induce mGlu1 receptor-stimulated neuroprotection. On the other hand, endogenous compounds including glutamate, aspartate, cysteic acid, cysteine sulfinic acid, and homocysteic acid stimulate both responses. These results show that some commonly used mGlu1 receptor ligands are biased agonists, stimulating only a fraction of mGlu1 receptor-mediated responses in neurons. This emphasizes the importance of utilizing multiple agonists and assays when studying GPCR function.
Asunto(s)
Cerebelo/citología , Neuronas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Animales Recién Nacidos , Arrestinas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Ácido Glutámico/farmacología , Hidrólisis/efectos de los fármacos , Neuronas/efectos de los fármacos , Fosfatidilinositoles/metabolismo , Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , beta-ArrestinasRESUMEN
The metabotropic glutamate 1 (mGlu1) receptor has emerged as a novel target for the treatment of metastatic melanoma and various other cancers. Our laboratory has demonstrated that a selective, non-competitive mGlu1 receptor antagonist slows human melanoma growth in vitro and in vivo. In this study, we sought to determine if the activation of a canonical G protein-dependent signal transduction cascade, which is often used as an output of mGlu1 receptor activity in neuronal cells, correlated with mGlu1 receptor-mediated melanoma cell viability. Glutamate, the endogenous ligand of mGlu1 receptors, significantly increased melanoma cell viability, but did not stimulate phosphoinositide (PI) hydrolysis in several human melanoma cell lines. In contrast, melanoma cell viability was not increased by quisqualate, a highly potent mGlu1 receptor agonist, or DHPG, a selective group I mGlu receptor agonist. Similarly to glutamate, quisqualate also failed to stimulate PI hydrolysis in mGlu1 receptor-expressing melanoma cells. These results suggest that the canonical G protein-dependent signal transduction cascade is not coupled to mGlu1 receptors in all human melanoma cells. On the other hand, dynamin inhibition selectively decreased viability of mGlu1 receptor-expressing melanoma cells, suggesting that a mechanism requiring internalization may control melanoma cell viability. Taken together, these data demonstrate that the approaches commonly used to study mGlu1 receptor function and signaling in other systems may be inappropriate for studying mGlu1 receptor-mediated melanoma cell viability.
Asunto(s)
Melanoma/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Adenosina Trifosfato/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Humanos , Ionomicina/farmacología , Ácido Quiscuálico/farmacología , Receptores de Glutamato Metabotrópico/genética , Sesquiterpenos/farmacología , Sesquiterpenos de Guayano , Transducción de SeñalRESUMEN
Glutamate is the major excitatory neurotransmitter in the CNS and its effects on neurons are dependent on the type and composition of glutamate receptors with which it interacts. In this study, the protein expression levels of several ionotropic glutamate receptor subunits (N-methyl-D-aspartate (NMDA) subunits NR1, NR2A, NR2B, and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor subunits GluR1, GluR2, GluR4) were quantified in particulate preparations from rat spinal cord at various ages after birth. We found that all six subunits showed high expression in the early postnatal period, followed by a subsequent decline as the rats matured to adults. The levels of two subunits (NR2A and GluR4) were found to initially increase during the first postnatal week prior to the decline to adult levels. The high levels of expression observed of these subunits in the early postnatal period may have implications for mechanisms of neural injury and cell death in the immature nervous system that involve cation influx through ionotropic glutamate receptors.
Asunto(s)
Diferenciación Celular/fisiología , Ácido Glutámico/metabolismo , Neuronas/metabolismo , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Médula Espinal/crecimiento & desarrollo , Médula Espinal/metabolismo , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Regulación hacia Abajo/fisiología , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Transmisión Sináptica/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
To investigate the possibility that glutamate receptor levels in the spinal cord are altered following injury to young rats, we used a previously characterized model of spinal cord contusion that produces a reliable injury in rats at postnatal day 14-15. Quantitative Western blot analysis was used to measure relative amounts of protein for several glutamate receptor subunits acutely (24 h) and chronically (28 days) after spinal cord injury (SCI). Acutely after injury significant decreases were observed in the GluR1, GluR2, and GluR4 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptor, and the NR2A and NR2B subunits, but not the NR1 subunit, of the N-methyl-d-aspartate (NMDA) receptor. However, 28 days after injury only one subunit (GluR4) was shown to be altered. These widespread changes that occur acutely in receptor subunit expression may be an attempt to protect cells from glutamate-induced death. The injured spinal cord in these young animals, however, appears to have the capacity to regulate receptor subunit levels to normal within a month of injury.
Asunto(s)
Receptores AMPA/biosíntesis , Receptores de N-Metil-D-Aspartato/biosíntesis , Traumatismos de la Médula Espinal/metabolismo , Factores de Edad , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Subunidades de Proteína/biosíntesis , Ratas , Ratas Sprague-Dawley , Recuperación de la Función , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
A key facet of professional development is the formation of professional identity. At its most basic level, professional identity for a scientist centers on mastery of a discipline and the development of research skills during doctoral training. To develop a broader understanding of professional identity in the context of doctoral training, the Carnegie Initiative on the Doctorate (CID) ran a multi-institutional study from 2001 to 2005. A key outcome of the CID was the development of the concept of 'stewards of the discipline'. The Interdisciplinary Program in Neuroscience (IPN) at Georgetown University participated in CID from 2003 to 2005. Here, we describe the IPN and highlight the programmatic developments resulting from participation in the CID. In particular, we emphasize programmatic activities that are designed to promote professional skills in parallel with scientific development. We describe activities in the domains of leadership, communication, teaching, public outreach, ethics, collaboration, and mentorship. Finally, we provide data that demonstrate that traditional metrics of academic success are not adversely affected by the inclusion of professional development activities in the curricula. By incorporating these seven 'professional development' activities into the required coursework and dissertation research experience, the IPN motivates students to become stewards of the discipline.