RESUMEN
Childhood stress and trauma are associated with substance use disorders in adulthood, but the neurological changes that confer increased vulnerability are largely unknown. In this study, maternal separation (MS) stress, restricted to the pre-weaning period, was used as a model to study mechanisms of protracted effects of childhood stress/traumatic experiences on binge drinking and impulsivity. Using an operant self-administration model of binge drinking and a delay discounting assay to measure impulsive-like behavior, we report that early life stress due to MS facilitated acquisition of binge drinking and impulsivity during adulthood in rats. Previous studies have shown heightened levels of corticotropin releasing factor (CRF) after MS, and here, we add that MS increased expression levels of GABA(A) α2 subunit in central stress circuits. To investigate the precise role of these circuits in regulating impulsivity and binge drinking, the CRF1 receptor antagonist antalarmin and the novel GABA(A) α2 subunit ligand 3-PBC were infused into the central amygdala (CeA) and medial prefrontal cortex (mPFC). Antalarmin and 3-PBC at each site markedly reduced impulsivity and produced profound reductions on binge-motivated alcohol drinking, without altering responding for sucrose. Furthermore, whole-cell patch-clamp studies showed that low concentrations of 3-PBC directly reversed the effect of relatively high concentrations of ethanol on α2ß3γ2 GABA(A) receptors, by a benzodiazepine site-independent mechanism. Together, our data provide strong evidence that maternal separation, i.e. early life stress, is a risk factor for binge drinking, and is linked to impulsivity, another key risk factor for excessive alcohol drinking. We further show that pharmacological manipulation of CRF and GABA receptor signaling is effective to reverse binge drinking and impulsive-like behavior in MS rats. These results provide novel insights into the role of the brain stress systems in the development of impulsivity and excessive alcohol consumption.
Asunto(s)
Consumo de Bebidas Alcohólicas/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Conducta Impulsiva/fisiología , Privación Materna , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Receptores de GABA-A/metabolismo , Estrés Psicológico/metabolismo , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Condicionamiento Operante/efectos de los fármacos , Etanol/administración & dosificación , Femenino , Masculino , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Pirimidinas/farmacología , Pirroles/farmacología , Ratas , Ratas Sprague-Dawley , Factores de Riesgo , Autoadministración , Vitamina B 12/análogos & derivadosRESUMEN
In this study we synthesized four different (18)F-labeling precursors for the visualization of the monoamino oxidase A using harmol derivatives. Whereas two are for prosthetic group labeling using [(18)F]fluoro-d2-methyl tosylate and 2-[(18)F]fluoroethyl-tosylate, the other three precursors are for direct nucleophilic (18)F-labeling. Additionally the corresponding reference compounds were synthesized. The syntheses of [(18)F]fluoro-d2-methyl-harmol and 2-[(18)F]fluoroethyl-harmol were carried out using harmol as starting material. For direct nucleophilic (18)F-labeling of the tracers carrying oligoethyled spacers (PEG), a toluenesulfonyl leaving group was employed. The radiolabeling, purification and formulation for each tracer was optimized and evaluated in vitro and in vivo. Stability tests in human serum showed that all tracers were stable over the observation period of 60 min. µPET studies using of the synthesized tracers revealed that the tracers carrying PEG spacers showed no sufficient brain uptake. Consequently, the (18)F-fuoro alkylated tracers [(18)F]fluoro-d2-methyl-harmol and 2-[(18)F]fluoroethyl-harmol were further evaluated showing SUVs in the brain of 1.0±0.2 g/mL and 3.4±0.5 g/mL after 45 min, respectively. In blockade studies the selectivity and specificity of both tracers were demonstrated. However, for [(18)F]fluoro-d2-methyl-harmol a rapid washout from the brain was also observed. In vitro binding assays revealed that 2-[(18)F]fluoroethyl-harmol (IC50=0.54±0.06 nM) has a higher affinity than the (18)F-fluoro-d2-methylated ligand (IC50=12.2±0.6 nM), making 2-[(18)F]fluoroethyl-harmol superior to the other evaluated compounds and a promising tracer for PET imaging of the MAO A.
Asunto(s)
Carbolinas/química , Radioisótopos de Flúor/química , Monoaminooxidasa/química , Animales , Carbolinas/sangre , Carbolinas/metabolismo , Estabilidad de Medicamentos , Humanos , Técnicas In Vitro , Marcaje Isotópico , Monoaminooxidasa/metabolismo , Tomografía de Emisión de Positrones/métodos , Radiofármacos/sangre , Radiofármacos/síntesis química , Radiofármacos/química , Ratas , Ratas Sprague-DawleyRESUMEN
Phencyclidine (PCP) and ketamine are dissociative anesthetics capable of inducing analgesia, psychomimetic behavior, and a catatonic state of unconsciousness. Despite broad similarities, there are notable differences between the clinical actions of ketamine and PCP. Ketamine has a lower incidence of adverse effects and generally produces greater CNS depression than PCP. Both noncompetitively inhibit NMDA receptors, yet there is little evidence that these drugs affect GABA(A) receptors, the primary target of most anesthetics. alpha6beta2/3delta receptors are subtypes of the GABA(A) receptor family and are abundantly expressed in granular neurons within the adult cerebellum. Here, using an oocyte expression system, we show that at anesthetically relevant concentrations, ketamine, but not PCP, modulates alpha6beta2delta and alpha6beta3delta receptors. Additionally, at higher concentrations, ketamine directly activates these GABA(A) receptors. Comparatively, dizocilpine (MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate]), a potent noncompetitive antagonist of NMDA receptors that is structurally unrelated to PCP, did not produce any effect on alpha6beta2delta receptors. Of the recombinant GABA(A) receptor subtypes examined (alpha1beta2, alpha1beta2gamma2, alpha1beta2delta, alpha4beta2gamma2, alpha4beta2delta, alpha6beta2gamma2, alpha6beta2delta, and alpha6beta3delta), the actions of ketamine were unique to alpha6beta2delta and alpha6beta3delta receptors. In dissociated granule neurons and cerebellar slice recordings, ketamine potentiated the GABAergic conductance arising from alpha6-containing GABA(A) receptors, whereas PCP showed no effect. Furthermore, ketamine potentiation was absent in cerebellar granule neurons from transgenic functionally null alpha6(-/-) and delta(-/-)mice. These findings suggest that the higher CNS depressant level achieved by ketamine may be the result of its selective actions on alpha6beta2/3delta receptors.
Asunto(s)
Corteza Cerebelosa/efectos de los fármacos , Ketamina/farmacología , Neuronas/efectos de los fármacos , Fenciclidina/farmacología , Receptores de GABA-A/efectos de los fármacos , Anestésicos Disociativos/farmacología , Animales , Células Cultivadas , Corteza Cerebelosa/metabolismo , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas/metabolismo , Oocitos , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Xenopus laevisRESUMEN
The nonsteroidal anti-inflammatory drug (NSAID) niflumic acid, a fenamate in structure, has many molecular targets, one of them being specific subtypes of the main inhibitory ligand-gated anion channel, the GABAA receptor. Here, we report on the effects of other fenamates and other classes of NSAIDs on brain picrotoxinin-sensitive GABAA receptors, using an autoradiographic assay with [35S]TBPS as a ligand on mouse brain sections. We found that the other fenamates studied (flufenamic acid, meclofenamic acid, mefenamic acid and tolfenamic acid) affected the autoradiographic signal at low micromolar concentrations in a facilitatory-like allosteric fashion, i.e., without having affinity to the [35S]TBPS binding site. Unlike niflumic acid that shows clear preference for inhibiting cerebellar granule cell layer GABAA receptors, the other fenamates showed little brain regional selectivity, indicating that their actions are not receptor-subtype selective. Of the non-fenamate NSAIDs studied at 100⯵M concentration, diclofenac induced the greatest inhibition of the binding, which is not surprising as it has close structural similarity with the potent fenamate meclofenamic acid. Using two-electrode voltage-clamp assays on Xenopus oocytes, the effect of niflumic acid was found to be dependent on the ß subunit variant and the presence of γ2 subunit in rat recombinant α1ß and α1ßγ2 GABAA receptors, with the ß1 allowing the niflumic acid inhibition and ß3 the stimulation of the receptor-mediated currents. In summary, the fenamate NSAIDs constitute an interesting class of compounds that could be used for development of potent GABAA receptor allosteric agonists with other targets to moderate inflammation, pain and associated anxiety/depression.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Fenamatos/farmacología , Receptores de GABA-A/metabolismo , Animales , Antiinflamatorios no Esteroideos/metabolismo , Fenómenos Electrofisiológicos/efectos de los fármacos , Fenamatos/metabolismo , Masculino , RatasRESUMEN
The α4/6ßδ-containing GABAA receptors are involved in a number of brain diseases. Despite the potential of a δ-selective imaging agent, no PET radioligand is currently available for in vivo imaging. Here, we report the characterization of DS2OMe (1) as a candidate radiotracer, 11C-labeling, and subsequent evaluation of [11C]DS2OMe in a domestic pig as a PET radioligand for visualization of the δ-containing GABAA receptors.
RESUMEN
The behavioral and functional significance of the extrasynaptic inhibitory GABA(A) receptors in the brain is still poorly known. We used a transgenic mouse line expressing the GABA(A) receptor alpha6 subunit gene in the forebrain under the Thy-1.2 promoter (Thy1alpha6) mice ectopically expressing alpha6 subunits especially in the hippocampus to study how extrasynaptically enriched alphabeta(gamma2)-type receptors alter animal behavior and receptor responses. In these mice extrasynaptic alpha6beta receptors make up about 10% of the hippocampal GABA(A) receptors resulting in imbalance between synaptic and extrasynaptic inhibition. The synthetic GABA-site competitive agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; 3 mg/kg) induced remarkable anxiolytic-like response in the light : dark exploration and elevated plus-maze tests in Thy1alpha6 mice, while being almost inactive in wild-type mice. The transgenic mice also lost quicker and for longer time their righting reflex after 25 mg/kg gaboxadol than wild-type mice. In hippocampal sections of Thy1alpha6 mice, the alpha6beta receptors could be visualized autoradiographically by interactions between gaboxadol and GABA via [(35)S]TBPS binding to the GABA(A) receptor ionophore. Gaboxadol inhibition of the binding could be partially prevented by GABA. Electrophysiology of recombinant GABA(A) receptors revealed that GABA was a partial agonist at alpha6beta3 and alpha6beta3delta receptors, but a full agonist at alpha6beta3gamma2 receptors when compared with gaboxadol. The results suggest strong behavioral effects via selective pharmacological activation of enriched extrasynaptic alphabeta GABA(A) receptors, and the mouse model represents an example of the functional consequences of altered balance between extrasynaptic and synaptic inhibition.
Asunto(s)
Conducta Animal/efectos de los fármacos , Agonistas del GABA/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Isoxazoles/farmacología , Receptores de GABA-A/metabolismo , Animales , Autorradiografía/métodos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Adaptación a la Oscuridad/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Conducta Exploratoria/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Unión Proteica/efectos de los fármacos , Subunidades de Proteína/metabolismo , Tiempo de Reacción/efectos de los fármacos , Receptores de GABA-A/genética , Reflejo/efectos de los fármacos , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Transfección/métodos , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Radiochemical labeling of MDL 105725 using the secondary labeling precursor 2-[(18)F]fluoroethyltosylate ([(18)F]FETos) was carried out in yields of approximately 90% synthesizing [(18)F]MHMZ in a specific activity of approximately 50MBq/nmol with a starting activity of approximately 3GBq. Overall radiochemical yield including [(18)F]FETos synthon synthesis, [(18)F]fluoroalkylation and preparing the injectable [(18)F]MHMZ solution was 42% within a synthesis time of approximately 100 min. The novel compound showed excellent specific binding to the 5-HT(2A) receptor (K(i)=9.0 nM) in vitro and promising in vivo characteristics.
Asunto(s)
Radioisótopos de Flúor/química , Fluorobencenos/síntesis química , Piperidinas/síntesis química , Radiofármacos/síntesis química , Antagonistas del Receptor de Serotonina 5-HT2 , Antagonistas de la Serotonina/síntesis química , Animales , Unión Competitiva , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Fluorobencenos/química , Marcaje Isotópico , Ketanserina/análogos & derivados , Ketanserina/farmacocinética , Cinética , Piperidinas/química , Ensayo de Unión Radioligante , Cintigrafía , Radiofármacos/farmacocinética , Ratas , Receptor de Serotonina 5-HT2A/metabolismo , Antagonistas de la Serotonina/farmacocinéticaRESUMEN
δ-Selective compounds 1 and 2 (DS1, compound 22; DS2, compound 16) were introduced as functionally selective modulators of δ-containing GABA type A receptors (GABAAR). In our hands, [3H]EBOB-binding experiments with recombinant GABAAR and compound 22 showed no proof of δ-selectivity, although there was a minimally higher preference for the α4ß3δ and α6ß2/3δ receptors with respect to potency. In order to delineate the structural determinants of δ preferences, we synthesized 25 derivatives of DS1 and DS2, and investigated their structure-activity relationships (SAR). Four of our derivatives showed selectivity for α6ß3δ receptors (29, 38, 39, and 41). For all of them, the major factors that distinguished them from compound 22 were variations at the para-positions of their benzamide groups. However, two compounds (29 and 39), when tested in the presence of GABA, revealed effects at several additional GABAAR. The newly synthesized compounds will still serve as useful tools to investigate α6ß3δ receptors.
Asunto(s)
Antagonistas de Receptores de GABA-A/química , Imidazoles/metabolismo , Piridinas/metabolismo , Receptores de GABA-A/metabolismo , Humanos , Imidazoles/química , Imidazoles/farmacología , Concentración 50 Inhibidora , Subunidades de Proteína/metabolismo , Piridinas/química , Piridinas/farmacología , Relación Estructura-ActividadRESUMEN
A chemically heterogeneous group of compounds acts at the benzodiazepine (BZ) recognition site of the diverse gamma-aminobutyric acid type A (GABA(A)) receptor complexes which can assemble from more than 16 known subunits. Most 1,4-BZs like diazepam recognize all GABA(A)/BZ receptors containing the alpha1-3 or alpha5 together with any beta and the gamma2 subunit. Other compounds differentiate less, e.g. Ro15-4513, that additionally recognizes alpha4- and a6-containing receptors, or differentiate more, e.g. zolpidem, that recognizes preferentially alpha1-containing receptors. Here we describe the functional properties of 1-(4-chloro-phenyl)-4-piperidin-1-yl-1,5-dihydro-imidazol-2-on (ELB139) in the presence and absence of the BZ receptor antagonist flumazenil (Ro15-1788) on recombinant alphaibeta2gamma2 (i=1-5) receptor subtypes expressed in HEK 293 cells. The properties were measured with the whole-cell variation of the patch-clamp technique and compared to those of diazepam. Like the latter, ELB139 did not potentiate GABA-induced currents in alpha4-containing receptors, but it displays functional subtype specificity between alpha1, alpha2, alpha3, and alpha5beta2gamma2 receptors with highest potency in alpha3-containing receptors but highest efficacy in alpha1- or alpha2-containing receptors, respectively. ELB139 acted as a partial agonist on these receptor subtypes reaching 40-50% of the efficacy of diazepam.
Asunto(s)
Ansiolíticos/farmacología , Diazepam/farmacología , Imidazoles/farmacología , Potenciales de la Membrana/efectos de los fármacos , Piperidinas/farmacología , Receptores de GABA-A/fisiología , Animales , Línea Celular Transformada , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Agonistas de Receptores de GABA-A , Humanos , Técnicas de Placa-Clamp/métodos , Ratas , Receptores de GABA-A/química , Transfección/métodosRESUMEN
In rodent models, gamma-aminobutyric acid A (GABAA) receptors with the alpha6 and delta subunits, expressed in the cerebellar and cochlear nucleus granule cells, have been linked to ethanol sensitivity and voluntary ethanol drinking. Here, we review the findings. When considering both in vivo contributions and data on cloned receptors, the evidence for direct participation of the alpha6-containing receptors to increased ethanol sensitivity is poor. The alpha6 subunit-knockout mouse lines do not have any changed sensitivity to ethanol, although these mice do display increased benzodiazepine sensitivity. However, in general the compensations occurring in knockout mice (regardless of which particular gene is knocked out) tend to fog interpretations of drug actions at the systems level. For example, the alpha6 knockout mice have increased TASK-1 channel expression in their cerebellar granule cells, which could influence sensitivity to ethanol in the opposite direction to that obtained with the alpha6 knockouts. Indeed, TASK-1 knockout mice are more impaired than wild types in motor skills when given ethanol; this might explain why GABAA receptor alpha6 knockout mice have unchanged ethanol sensitivities. As an alternative to studying knockout mice, we examined the claimed delta subunit-dependent/gamma2 subunit-independent ethanol/[3H]Ro 15-4513 binding sites on GABAA receptors. We looked at [3H]Ro 15-4513 binding in HEK 293 cell membrane homogenates containing rat recombinant alpha6/4beta3delta receptors and in mouse brain sections. Specific high-affinity [3H]Ro 15-4513 binding could not be detected under any conditions to the recombinant receptors or to the cerebellar sections of gamma2(F77I) knockin mice, nor was this binding to brain sections of wild-type C57BL/6 inhibited by 1-100 mM ethanol. Since ethanol may act on many receptor and channel protein targets in neuronal membranes, we consider the alpha6 (and alpha4) subunit-containing GABAA receptors unlikely to be directly responsible for any major part of ethanol's actions. Therefore, we finish the review by discussing more generally alcohol and GABAA receptors and by suggesting potential future directions for this research.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Receptores de GABA-A/efectos de los fármacos , Animales , Azidas/metabolismo , Azidas/farmacología , Benzodiazepinas/metabolismo , Benzodiazepinas/farmacología , Unión Competitiva/efectos de los fármacos , Depresores del Sistema Nervioso Central/antagonistas & inhibidores , Tolerancia a Medicamentos , Etanol/antagonistas & inhibidores , Humanos , Ratones , Ratas , Receptores de GABA-A/genéticaRESUMEN
As a cellular bile acid sensor, farnesoid X receptor (FXR) participates in regulation of bile acid, lipid and glucose homeostasis, and liver protection. Clinical results have validated FXR as therapeutic target in hepatic and metabolic diseases. To date, potent FXR agonists share a negatively ionizable function that might compromise their pharmacokinetic distribution and behavior. Here we report the development and characterization of a high-affinity FXR modulator not comprising an acidic residue.
Asunto(s)
Imidazoles/farmacología , Piridinas/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Animales , Colesterol 7-alfa-Hidroxilasa/genética , Estabilidad de Medicamentos , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Imidazoles/síntesis química , Imidazoles/química , Imidazoles/metabolismo , Masculino , Proteínas de Transporte de Membrana/genética , Simulación del Acoplamiento Molecular , PPAR alfa/genética , Piridinas/síntesis química , Piridinas/química , Piridinas/metabolismo , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Relación Estructura-Actividad , ZolpidemRESUMEN
Neurotransmitter receptor systems have been the focus of intensive pharmacological research for more than 20 years for basic and applied scientific reasons, but only recently has there been a better understanding of their key features. One of these systems includes the type A receptor for the gamma-aminobutyric acid (GABA), which forms an integral anion channel from a pentameric subunit assembly and mediates most of the fast inhibitory neurotransmission in the adult vertebrate central nervous system. Up to now, depending on the definition, 16-19 mammalian subunits have been cloned and localized on different genes. Their assembly into proteins in a poorly defined stoichiometry forms the basis of functional and pharmacological GABA(A) receptor diversity, i.e. the receptor subtypes. The latter has been well documented in autoradiographic studies using ligands that label some of the receptors' various binding sites, corroborated by recombinant expression studies using the same tools. Significantly less heterogeneity has been found at the physiological level in native receptors, where the subunit combinations have been difficult to dissect. This review focuses on the characteristics, use and usefulness of various ligands and their binding sites to probe GABA(A) receptor properties and to gain insight into the biological function from fish to man and into evolutionary conserved GABA(A) receptor heterogeneity. We also summarize the properties of the novel mouse models created for the study of various brain functions and review the state-of-the-art imaging of brain GABA(A) receptors in various human neuropsychiatric conditions. The data indicate that the present ligands are only partly satisfactory tools and further ligands with subtype-selective properties are needed for imaging purposes and for confirming the behavioral and functional results of the studies presently carried out in gene-targeted mice with other species, including man.
Asunto(s)
Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Receptores de GABA-A , Secuencia de Aminoácidos , Animales , Interacciones Farmacológicas , Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Humanos , Datos de Secuencia Molecular , Receptores de GABA-A/fisiologíaRESUMEN
Endogenous neurosteroids and neuroactive steroids have potent and widespread actions on the brain via inhibitory GABAA receptors. In recombinant receptors and genetic mouse models their actions depend on the α, ß, and δ subunits of the receptor, especially on those that form extrasynaptic GABAA receptors responsible for non-synaptic (tonic) inhibition, but they also act on synaptically enriched γ2 subunit-containing receptors and even on αß binary receptors. Here we tested whether behavioral sensitivity to the neuroactive steroid agonist 5ß-pregnan-3α-ol-20-one is altered in genetically engineered mouse models that have deficient GABAA receptor-mediated synaptic inhibition in selected neuronal populations. Mouse lines with the GABAA receptor γ2 subunit gene selectively deleted either in parvalbumin-containing cells (including cerebellar Purkinje cells), cerebellar granule cells, or just in cerebellar Purkinje cells were trained on the accelerated rotating rod and then tested for motor impairment after cumulative intraperitoneal dosing of 5ß-pregnan-3α-ol-20-one. Motor-impairing effects of 5ß-pregnan-3α-ol-20-one were strongly increased in all three mouse models in which γ2 subunit-dependent synaptic GABAA responses in cerebellar neurons were genetically abolished. Furthermore, rescue of postsynaptic GABAA receptors in Purkinje cells normalized the effect of the steroid. Anxiolytic/explorative effects of the steroid in elevated plus maze and light:dark exploration tests in mice with Purkinje cell γ2 subunit inactivation were similar to those in control mice. The results suggest that, when the deletion of γ2 subunit has removed synaptic GABAA receptors from the specific cerebellar neuronal populations, the effects of neuroactive steroids solely on extrasynaptic αß or αßδ receptors lead to enhanced changes in the cerebellum-generated behavior.
RESUMEN
In this study, we investigate how neurosteroid sensitivity of GABA(A) receptors (GABA(A)Rs) is regulated. We examined this issue in neurons of the supraoptic nucleus (SON) of the rat and found that, during parturition, the GABA(A)Rs become insensitive to the neurosteroid allopregnanolone attributable to a shift in the balance between the activities of endogenous Ser/Thr phosphatase and PKC. In particular, a constitutive endogenous tone of oxytocin within the SON after parturition suppressed neurosteroid sensitivity of GABA(A)Rs via activation of PKC. Vice versa before parturition, during late pregnancy, application of exogenous oxytocin brings the GABA(A)Rs from a neurosteroid-sensitive mode toward a condition in which the receptors are not sensitive. This indicates that there may be an inverse causal relationship between the extent to which the GABA(A)R or one of its interacting proteins is phosphorylated and the neurosteroid sensitivity of the GABA(A)R. Neurosteroid sensitivity was not affected by changes in subunit composition of GABA(A)Rs known to occur concurrently in these cells.
Asunto(s)
Oxitocina/farmacología , Receptores de GABA-A/metabolismo , Esteroides/farmacología , Núcleo Supraóptico/metabolismo , Animales , Animales Recién Nacidos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Línea Celular , Femenino , Humanos , Riñón/citología , Riñón/metabolismo , Ligandos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Oocitos/metabolismo , Oxitocina/metabolismo , Técnicas de Placa-Clamp , Fosfoproteínas Fosfatasas/metabolismo , Embarazo , Pregnanolona/farmacología , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/genética , Núcleo Supraóptico/efectos de los fármacos , Transfección , Xenopus , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Irradiation resistance is a major obstacle of head and neck squamous cell carcinoma (HNSCC) therapy, limiting treatment success and patient survival. The aim of our experiments was to identify irradiation-regulated proteins as potential drug targets. Two established HNSCC cell lines (HNSCCUM-01T and HNSCCUM-02T) were treated with a single 8Gy (Gray) fraction of irradiation. Changes in cellular protein expression were studied after 24h by means of 2D-electrophoresis and MALDI-TOF-mass spectrometry. Ninety-four differentially expressed proteins were identified. The expression levels of four proteins were regulated similarly in both cell lines after irradiation treatment, i.e., GRP78, PRDX, ACTC, and the heterogeneous nuclear ribonucleoprotein K (hnRNPK), suggesting a relevant role during irradiation response. hnRNPK as a p53 interacting protein was verified by Western blotting and immunocytochemical staining as well as functionally analyzed. Knock-down by the use of siRNA resulted in only slightly reduced viability, however, migratory activity was strongly reduced. Combined application of siRNA against hnRNPK and irradiation reduced migration almost completely. We conclude that hnRNPK is potentially implicated in the radiogenic response of HNSCC. The inhibition of hnRNPK might reduce the metastasizing potential of HNSCC especially in combination with irradiation and suggest that this molecule should be further evaluated in this context. BIOLOGICAL SIGNIFICANCE: We showed completely impaired migration of irradiated hnRNPK-knock-out HNSCC cells, suggesting this molecule as a potential drug target in combined treatment schedules.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Movimiento Celular/efectos de la radiación , Rayos gamma , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Neoplasias de Cabeza y Cuello/metabolismo , Proteínas de Neoplasias/metabolismo , Ribonucleoproteínas/biosíntesis , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/radioterapia , Línea Celular Tumoral , Chaperón BiP del Retículo Endoplásmico , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/radioterapia , Ribonucleoproteína Heterogénea-Nuclear Grupo K , Humanos , Metástasis de la Neoplasia , ProteómicaRESUMEN
To image the possible alterations in brain regional GABAA receptor subtype properties in a genetic animal model of human anxiety, mice heterozygous for the deletion of GABAA receptor gamma2 subunit (gamma2+/-) were studied using ligand autoradiographic assays on brain cryostat sections. The [35S]TBPS binding assay was designed to reveal impaired GABA and channel site coupling shown to be more prominent in recombinant alpha1/6beta3 than in alpha1/2beta3gamma2 or beta2 subunit-containing GABAA receptors expressed in HEK 293 cells. Increased GABA-insensitive [35 S]TBPS binding in the gamma2+/- mouse brains was evident in the cerebral cortex and in subcortical regions, the alterations being regionally similar to the loss of gamma2 subnunit-dependent benzodiazepine (BZ) sites as revealed by [3H]Ro 15-4513 autoradiography. As the gamma2 subunit protein is needed for synaptic clustering of GABAA receptors, these results indicate that the extrasynaptic alphabeta3 receptors can be visualized in vitro as atypical GABA-insensitive [35S]TBPS binding sites. The results suggest that GABAAergic synaptic inhibition is widely decreased in the brains of anxiety-prone gamma2+/- mice, while extrasynaptic GABAA receptors are increased. These autoradiographic imaging findings further demonstrate the need to develop GABAA receptor subtype-selective in vivo ligands to aid in assessing the contributions of various subcellular receptor populations in anxious and other patient groups.
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Ansiedad/genética , Ansiedad/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de GABA/genética , Receptores de GABA/metabolismo , Sinapsis/metabolismo , Animales , Autorradiografía , Azidas/farmacología , Benzodiazepinas/farmacología , Femenino , Agonistas del GABA , Masculino , Ratones , Ratones Noqueados , Muscimol , Radioisótopos de Azufre , TransfecciónRESUMEN
The preservation of the neuronal circuitry in rat cerebellar slice cultures provides an advantage in monitoring the development and characterizing the pharmacology of GABA(A) receptor subtypes. Sprague-Dawley rats, 8-11 days of age, were decapitated, their cerebella were cut into 400-microm slices and transferred into culture dishes. Cell viability and organotypic cerebellar organization of the culture remained well preserved up to 3 weeks. Autoradiographic procedures were introduced in these advanced culture technique and employed [(3)H]Ro 15-4513 in the absence and presence of 10 microM diazepam to visualize all benzodiazepine (BZD) and diazepam-insensitive (DIS) binding sites, respectively. Since expression of the alpha6 subunit variant of the GABA(A)/BZD receptor is restricted to the cerebellar granule cells and the BZD receptor agonist diazepam has very low affinity for this subunit, changes in DIS [(3)H]Ro 15-4513 binding sites during cultivation time can be attributed to changes in alpha6 subunit expression. A time-dependent development of total and DIS [(3)H]Ro 15-4513 binding sites were observed in the culture with a trend towards an increase in GABA(A) receptor alpha6 subunit levels during the first week. These findings suggest that explant preparations can be used to examine morphological changes in rat cerebellar slices. In addition, these preparations can be utilized to study the pharmacological effects of GABA(A)/BZD selective drugs on postnatal development of GABA(A) receptors in rat cerebellum.
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Cerebelo/metabolismo , Receptores de GABA-A/metabolismo , Marcadores de Afinidad/metabolismo , Animales , Animales Recién Nacidos , Azidas/metabolismo , Benzodiazepinas/metabolismo , Sitios de Unión/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Cerebelo/citología , Técnicas de Cultivo/métodos , Masculino , Especificidad de Órganos , Ratas , Ratas Sprague-DawleyRESUMEN
Ethyl alcohol (ethanol) has many molecular targets in the nervous system, its potency at these sites being low compared to those of sedative drugs. This has made it difficult to discover ethanol's binding site(s). There are two putative binding sites at γ-aminobutyric acid (GABA) type A receptor subtypes for the proposed ethanol antagonist Ro 15-4513, the established γ2 subunit-dependent benzodiazepine site and the recently reported δ subunit-dependent Ro 15-4513/ethanol binding site. Here, we aimed at clarifying the in vivo role of Ro 15-4513 at these two sites. We found that the antagonism of ethanol actions by Ro 15-4513 in wildtype mice was dependent on the test: an open field test showed that light sedation induced by 1.5-1.8 g/kg ethanol was sensitive to Ro 15-4513, whereas several tests for ethanol-induced anxiolytic effects showed that the ethanol-induced effects were insensitive to Ro 15-4513. Antagonism of ethanol-induced sedation by Ro 15-4513 was unaffected in GABA(A) receptor δ subunit knockout mice. By contrast, when testing the GABA(A) receptor γ2 subunit F77I knock-in mouse line (γ2I77 mice) with its strongly reduced affinity of the benzodiazepine sites for Ro 15-4513, we found that the ethanol-induced sedation was no longer antagonized by Ro 15-4513. Indeed, γ2I77 mice had only a small proportion of high-affinity binding of [(3)H]Ro 15-4513 left as compared to wildtype mice, especially in the caudate-putamen and septal areas, but these residual sites are apparently not involved in ethanol antagonism. In conclusion, we found that Ro 15-4513 abolished the sedative effect of ethanol by an action on γ2 subunit-dependent benzodiazepine sites.
RESUMEN
The potent sedative-hypnotic zolpidem and the convulsant methyl-6,7-dimethoxy-4-ethyl-ß-carboline-3-carboxylate (DMCM) act primarily by binding to the benzodiazepine site of the main inhibitory neurotransmitter receptor, the pentameric γ-aminobutyric acid type A receptor (GABA(A)). This binding depends critically on the wild-type F77 residue of the GABA(A) receptor γ2 subunit. Mice with γ2 subunit F77I point mutation (γ2I77 mouse line) lose the high-affinity nanomolar binding of these ligands as well as their most robust behavioral actions at low doses. Interestingly, the γ2I77 mice offer a tool to study the actions of these substances mediated via other possible binding sites of the GABA(A) receptor. In ligand autoradiographic experiments, we discovered in γ2I77 mouse brain sections a significant amount of residual non-γ2 subunit-dependent benzodiazepine site binding enriched to the striatum and septum. Zolpidem only weakly affected this residual binding at micromolar concentrations, and only a high zolpidem dose (≥ 40 mg/kg) caused sedation and deficits in motor coordination in γ2I77 mice. DMCM had an agonistic action through a secondary, low-affinity non-benzodiazepine binding site of the GABA(A) receptor in the forebrain of γ2I77 mice, and this drug also fully displaced the residual benzodiazepine-site labeling. In behavioral tests, a high dose (20mg/kg) of DMCM was sedative and modulated fear learning. DMCM, but not zolpidem, acted as an agonist in recombinant GABA(A) α1/6ß3 receptors studied using ligand binding and electrophysiological assays. Our results highlight the less well-known actions of high doses of DMCM and zolpidem that are not mediated via the γ2 subunit-containing benzodiazepine site of the GABA(A) receptor.
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Benzodiazepinas/metabolismo , Carbolinas/metabolismo , Carbolinas/farmacología , Piridinas/metabolismo , Piridinas/farmacología , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Animales , Autorradiografía , Azidas/farmacología , Conducta Animal/efectos de los fármacos , Benzodiazepinas/farmacología , Sitios de Unión/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Convulsivantes/metabolismo , Convulsivantes/farmacología , Femenino , Células HEK293 , Humanos , Hipnóticos y Sedantes/metabolismo , Hipnóticos y Sedantes/farmacología , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica/efectos de los fármacos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ZolpidemRESUMEN
INTRODUCTION: The serotonergic system, especially the 5-HT2A receptor, is involved in various diseases and conditions. It is a very interesting target for medicinal applications. METHODS: Two novel 5-HT2A tracers, namely, [(18)F]DD-1 and the enantiomeric pure (R)-[(18)F]MH.MZ, were radiolabeled by (18)F-fluoroalkylation of the corresponding desmethyl analogue. In vitro binding autoradiography on rat brain slices was performed to test the affinity and selectivity of these tracers. Moreover, first microPET experiments of (R)-[(18)F]MH.MZ were carried out in Sprague-Dawley rats. RESULTS: [(18)F]DD-1 (K(i)=3.23 nM) and (R)-[(18)F]MH.MZ (K(i)=0.72 nM) were (18)F-fluoroalkylated by the secondary synthon [(18)F]FETos in a radiochemical yield (RCY) of >70%. The final formulation for both tracers took no longer than 100 min with an overall RCY of approximately 40%. It provided [(18)F]tracers with a purity >96% and a typical specific activity of 25-35 GBq/mumol. Autoradiographic images of (R)-[(18)F]MH.MZ (5) and [(18)F]DD-1 (4) showed excellent visualization and selectivity of the 5-HT2A receptor for (R)-[(18)F]MH.MZ and less specific binding for [(18)F]DD-1. The binding potential (BP) of (R)-[(18)F]MH.MZ was determined to be 2.6 in the frontal cortex and 2.2 in the cortex (n=4), whereas the cortex-to-cerebellum ratio was determined to be 3.2 at steady state (n=4). Cortex-to-cerebellum ratios of (R)-[(18)F]MH.MZ were almost twice as much as compared with the racemic [(18)F]MH.MZ. Thereby, equal levels of specific activities were used. High uptake could be demonstrated in cortex regions. CONCLUSION: Labeling of both novel tracers was carried out in high RCY. Autoradiography revealed (R)-[(18)F]MH.MZ as a very selective and affine 5-HT2A tracer (K(i)=0.72 nM), whereas [(18)F]DD-1 showed no reasonable distribution pattern on autoradiographic sections. Moreover, results from microPET scans of (R)-[(18)F]MH.MZ hint on improved molecular imaging characteristics compared with those of [(18)F]MH.MZ. Therefore, (R)-[(18)F]MH.MZ appears to be a highly potent and selective serotonergic PET ligand in small animals.