RESUMEN
BACKGROUND: To assess GABAA receptor subtypes involved in benzodiazepine tolerance and dependence, we evaluated the ability of subtype-selective and non-selective ligands to substitute for (i.e., produce "cross-tolerance") or precipitate withdrawal during chronic alprazolam treatment. METHODS: Four female rhesus monkeys (Macaca mulatta) were implanted with chronic intravenous catheters and administered alprazolam (1.0 mg/kg every 4 h). Following 14+ days of chronic alprazolam, acute administration of selected doses of non-selective and subtype-selective ligands were substituted for, or administered with, alprazolam, followed by quantitative behavioral observations. The ligands included alprazolam and midazolam (positive modulators, non-selective), zolpidem (positive modulator, preferential affinity for α1-containing GABAA receptors), HZ-166 (positive modulator, preferential efficacy at α2- and α3-containing GABAA receptors), and ßCCT (antagonist, preferential affinity for α1-containing GABAA receptors). RESULTS: Acutely, alprazolam and midazolam both induced observable ataxia along with a mild form of sedation referred to as "rest/sleep posture" at a lower dose (0.1 mg/kg, i.v.), whereas at a higher dose (1.0 mg/kg, i.v.), induced deep sedation and observable ataxia. With chronic alprazolam treatment, observable ataxia and deep sedation were reduced significantly, whereas rest/sleep posture was unchanged or emerged. Zolpidem showed a similar pattern of effects, whereas no behaviors engendered by HZ-166 were changed by chronic alprazolam. Administration of ßCCT, but not HZ-166, resulted in significant withdrawal signs. CONCLUSIONS: These results are consistent with a role for α1-containing GABAA receptor subtypes in tolerance and dependence observed with chronic alprazolam, although other receptors may be involved in the withdrawal syndrome.
Asunto(s)
Alprazolam , Tolerancia a Medicamentos , Receptores de GABA-A , Síndrome de Abstinencia a Sustancias , Alprazolam/farmacología , Animales , Benzodiazepinas , Femenino , Macaca mulatta , Midazolam/farmacología , Receptores de GABA-A/clasificación , ZolpidemRESUMEN
The modulation of neuronal cell firing is mediated by the release of the neurotransmitter GABA (γ-aminobuytric acid), which binds to two major families of receptors. The ionotropic GABAA receptors (GABAARs) are composed of five distinct subunits that vary in expression by brain region and cell type. The action of GABA on GABAARs is modulated by a variety of clinically and pharmacologically important drugs such as benzodiazepines and alcohol. Exposure to and abuse of these substances disrupts homeostasis and induces plasticity in GABAergic neurotransmission, often via the regulation of receptor expression. Here, we review the regulation of GABAAR subunit expression in adaptive and pathological plasticity, with a focus on substance use. We examine the factors influencing the expression of GABAAR subunit genes including the regulation of the 5' and 3' untranslated regions, variations in DNA methylation, immediate early genes and transcription factors that regulate subunit expression, translational and post-translational modifications, and other forms of receptor regulation beyond expression. Advancing our understanding of the factors regulating GABAAR subunit expression during adaptive plasticity, as well as during substance use and withdrawal will provide insight into the role of GABAergic signaling in substance use disorders, and contribute to the development of novel targeted therapies.
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Regulación de la Expresión Génica , Receptores de GABA-A/metabolismo , Trastornos Relacionados con Sustancias/patología , Transmisión Sináptica , Ácido gamma-Aminobutírico/metabolismo , Animales , Humanos , Subunidades de Proteína , Receptores de GABA-A/clasificación , Receptores de GABA-A/genética , Trastornos Relacionados con Sustancias/metabolismoRESUMEN
Altered expression of GABA receptors (GABAARs) has been implicated in neurological and psychiatric disorders, but limited information about region-specific GABAAR subunit expression in healthy human brains, heteromeric assembly of major isoforms, and their collective organization across healthy individuals, are major roadblocks to understanding their role in non-physiological states. Here, by using microarray and RNA-Seq datasets-from single cell nuclei to global brain expression-from the Allen Institute, we find that transcriptional expression of GABAAR subunits is anatomically organized according to their neurodevelopmental origin. The data show a combination of complementary and mutually-exclusive expression patterns that delineate major isoforms, and which is highly stereotypical across brains from control donors. We summarize the region-specific signature of GABAR subunits per subject and its variability in a control population sample that can be used as a reference for remodeling changes during homeostatic rearrangements of GABAAR subunits after physiological, pharmacological or pathological challenges.
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Encéfalo/metabolismo , Subunidades de Proteína/genética , Receptores de GABA-A/genética , Transcriptoma , Adulto , Amígdala del Cerebelo/anatomía & histología , Amígdala del Cerebelo/metabolismo , Encéfalo/anatomía & histología , Corteza Cerebral/anatomía & histología , Corteza Cerebral/metabolismo , Cuerpo Estriado/anatomía & histología , Cuerpo Estriado/metabolismo , Conjuntos de Datos como Asunto , Femenino , Perfilación de la Expresión Génica , Hipocampo/anatomía & histología , Hipocampo/metabolismo , Humanos , Hipotálamo/anatomía & histología , Hipotálamo/metabolismo , Masculino , Mesencéfalo/anatomía & histología , Mesencéfalo/metabolismo , Persona de Mediana Edad , Especificidad de Órganos , Filogenia , Subunidades de Proteína/clasificación , Subunidades de Proteína/metabolismo , Receptores de GABA-A/clasificación , Receptores de GABA-A/metabolismoRESUMEN
In the past 20 years we have learned a great deal about GABAA receptor (GABAAR) subtypes, and which behaviors are regulated or which drug effects are mediated by each subtype. However, the question of where GABAARs involved in specific drug effects and behaviors are located in the brain remains largely unanswered. We review here recent studies taking a circuit pharmacology approach to investigate the functions of GABAAR subtypes in specific brain circuits controlling fear, anxiety, learning, memory, reward, addiction, and stress-related behaviors. The findings of these studies highlight the complexity of brain inhibitory systems and the importance of taking a subtype-, circuit-, and neuronal population-specific approach to develop future therapeutic strategies using cell type-specific drug delivery.
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Benzodiazepinas/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Receptores de GABA-A/clasificación , Ácido gamma-Aminobutírico/farmacología , Animales , Agonistas de Receptores de GABA-A/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Humanos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Receptores de GABA-A/metabolismoRESUMEN
Neurosteroids are the principal endogenous modulators of the γ-Aminobutyric acid receptors (GABAARs), pentameric membrane-bound proteins that can be assembled from at least 19 subunits. In the most abundant GABAAR arrangement (α1ß2γ2), neurosteroids can potentiate the GABA action as well as produce a direct activation of the channel. The recent crystal structures of neurosteroids bound to α homopentameric GABAAR reveal binding to five equivalent sites. However, these results have been obtained using receptors that are not physiologically relevant, suggesting a need to investigate neurosteroid binding to heteropentameric receptors that exist in the central nervous system. In a previous work, we predicted the neurosteroid binding site by applying molecular modeling methods on the ß3 homopentamer. Here we construct a homology model of the transmembrane domain of the heteropentameric α1ß2γ2 receptor and then, by combining docking and molecular dynamics simulations, we analyzed neurosteroid binding. Results show that the five neurosteroid cavities are conserved in the α1ß2γ2 receptor and all of them are able to bind neurosteroids. Two different binding modes were detected depending on the identity of the residue at position 241 in the transmembrane helix 1. These theoretical findings provide microscopic insights into neurosteroid binding at the heteropentameric GABAAR. The existence of two classes of sites may be associated with how neurosteroids modulate GABAAR. Our finding would represent the essential first step to reach a comprehensive understanding of how these endogenous molecules regulate the central nervous system.
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Pregnanolona/química , Pregnanolona/metabolismo , Conformación Proteica , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Dominios Proteicos , Receptores de GABA-A/clasificación , Homología de SecuenciaRESUMEN
Gamma-aminobutyric acid (GABA) is an important neurotransmitter, but recent reports have revealed the expression of GABAergic components in peripheral, non-neural tissues. GABA administration induces natriuresis and lowers blood pressure, suggesting renal GABA targets. However, systematic evaluation of renal GABAergic components has not been reported. In this study, kidney cortices of Wistar-Kyoto rats (WKY) were used to assay for messenger RNAs of GABA-related molecules using RT-PCR. In WKY kidney cortex, GABAA receptor subunits, α1, ß3, δ, ε and π, in addition to both types of GABAB receptors, R1 and R2, and GABAC receptor ρ1 and ρ2 subunit mRNAs were detected. Kidney cortex also expressed mRNAs of glutamate decarboxylase (GAD) 65, GAD67, 4-aminobutyrate aminotransferase and GABA transporter, GAT2. Western blot and/or immunohistochemistry were performed for those molecules detected by RT-PCR. By immunofluorescent observation, co-staining of α1, ß3, and π subunits was observed mainly on the apical side of cortical tubules, and immunoblot of kidney protein precipitated with π subunit antibody revealed α1 and ß3 subunit co-assembly. This is the first report of GABAA receptor π subunit in the kidney. In summary, unique set of GABA receptor subunits and subtypes were found in rat kidney cortex. As GABA producing enzymes, transporters and degrading enzyme were also detected, a possible existence of local renal GABAergic system with an autocrine/paracrine mechanism is suggested.
Asunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Corteza Renal/metabolismo , Receptores de GABA-A/genética , Ácido gamma-Aminobutírico/biosíntesis , 4-Aminobutirato Transaminasa/genética , 4-Aminobutirato Transaminasa/metabolismo , Adulto , Animales , Femenino , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Expresión Génica , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Humanos , Hipertensión/genética , Hipertensión/metabolismo , Inmunohistoquímica , Masculino , Subunidades de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores de GABA-A/clasificación , Receptores de GABA-A/metabolismoRESUMEN
The GABAA receptors (GABAARs) play an important role in inhibitory transmission in the brain. The GABAARs could be identified using a medicinal chemistry approach to characterize with a series of chemical structural analogues, some identified in nature, some synthesized, to control the structural conformational rigidity/flexibility so as to define the 'receptor-specific' GABA agonist ligand structure. In addition to the isosteric site ligands, these ligand-gated chloride ion channel proteins exhibited modulation by several chemotypes of allosteric ligands, that help define structure and function. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABAARs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Also in the trans-membrane domain are allosteric modulatory ligand sites, mostly positive, for diverse chemotypes with general anesthetic efficacy, namely, the volatile and intravenous agents: barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are apparent endogenous positive allosteric modulators of GABAARs. These binding sites depend on the GABAAR heteropentameric subunit composition, i.e., subtypes. Two classes of pharmacologically very important allosteric modulatory ligand binding site reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site, and the low-dose ethanol site. The benzodiazepine site is specific for certain subunit combination subtypes, mainly synaptically localized. In contrast, the low-dose (high affinity) ethanol site(s) is found at a modified benzodiazepine site on different, extrasynaptic, subtypes.
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Receptores de GABA-A/clasificación , Sitio Alostérico , Animales , Benzodiazepinas/metabolismo , Humanos , Ligandos , Receptores de GABA-A/metabolismoRESUMEN
Migraine is a common neurological episodic disorder with a female-to-male prevalence 3- to 4-fold higher, suggesting a possible X-linked genetic component. Our aims were to assess the role of common variants of gamma-aminobutyric acid A receptor (GABAAR) genes, located in the X-chromosome, in migraine susceptibility and the possible interaction between them. An association study with 188 unrelated cases and 286 migraine-free controls age- and ethnic matched was performed. Twenty-three tagging SNPs were selected in three genes (GABRE, GABRA3 and GABRQ). Allelic, genotypic and haplotypic frequencies were compared between cases and controls. We also focused on gene-gene interactions. The AT genotype of rs3810651 of GABRQ gene was associated with an increased risk for migraine (OR: 4.07; 95% CI: 1.71-9.73, p=0.002), while the CT genotype of rs3902802 (OR: 0.41; 95% CI: 0.21-0.78, p=0.006) and GA genotype of rs2131190 of GABRA3 gene (OR: 0.53; 95% CI: 0.32-0.88, p=0.013) seem to be protective factors. All associations were found in the female group and maintained significance after Bonferroni correction. We also found three nominal associations in the allelic analyses although there were no significant results in the haplotypic analyses. Strikingly, we found strong interactions between six SNPs encoding for different subunits of GABAAR, all significant after permutation correction. To our knowledge, we show for the first time, the putative involvement of polymorphisms in GABAAR genes in migraine susceptibility and more importantly we unraveled a role for novel gene-gene interactions opening new perspectives for the development of more effective treatments.
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Predisposición Genética a la Enfermedad , Trastornos Migrañosos/genética , Receptores de GABA-A/genética , Adolescente , Adulto , Edad de Inicio , Alelos , Estudios de Casos y Controles , Niño , Preescolar , Análisis por Conglomerados , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Receptores de GABA-A/clasificación , Adulto JovenRESUMEN
Non-selective positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) are known to impair anterograde memory. The role of the various GABAAR subtypes in the memory-impairing effects of non-selective GABAAR PAMs has not been fully elucidated. The current study assessed, in rhesus monkeys, effects of modulation of α1, α2/3, and α5GABAARs on visual recognition and spatial working memory using delayed matching-to-sample (DMTS) and self-ordered spatial search (SOSS) procedures, respectively. The DMTS procedure (n=8) involved selecting a previously presented 'sample' image from a set of multiple images presented after a delay. The SOSS procedure (n=6) involved touching a number of boxes without repeats. The non-selective GABAAR PAM triazolam and the α1GABAA preferential PAMS zolpidem and zaleplon reduced accuracy in both procedures, whereas the α5GABAA preferential PAMs SH-053-2'F-R-CH3 and SH-053-2'F-S-CH3, and the α2/3GABAA preferential PAM TPA023B were without effects on accuracy or trial completion. The low-efficacy α5GABAAR negative allosteric modulator (NAM) PWZ-029 slightly increased only DMTS accuracy, whereas the high-efficacy α5GABAAR NAMs RY-23 and RY-24 did not affect accuracy under either procedure. Finally, the slopes of the accuracy dose-effect curves for triazolam, zolpidem, and zaleplon increased with box number in the SOSS procedure, but were equivalent across DMTS delays. The present results suggest that (1) α1GABAARs, compared with α2/3 and α5GABAARs, are primarily involved in the impairment, by non-selective GABAAR PAMs, of visual recognition and visuospatial working memory in nonhuman primates; and (2) relative cognitive impairment produced by positive modulation of GABAARs increases with number of locations to be remembered, but not with the delay for remembering.
Asunto(s)
Memoria a Corto Plazo/efectos de los fármacos , Receptores de GABA-A/clasificación , Receptores de GABA-A/metabolismo , Reconocimiento en Psicología/efectos de los fármacos , Acetamidas/farmacología , Regulación Alostérica , Animales , Benzodiazepinas/farmacología , Condicionamiento Operante/efectos de los fármacos , Diazepam/análogos & derivados , Diazepam/farmacología , Relación Dosis-Respuesta a Droga , Imidazoles/farmacología , Macaca mulatta , Masculino , Piridazinas/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Receptores de GABA-A/química , Triazolam/farmacología , Triazoles/farmacología , ZolpidemRESUMEN
The present studies evaluated the role of α1 and α5 subunit-containing GABAA receptors (α1GABAA and α5GABAA receptors, respectively) in the ability of benzodiazepine (BZ)-type drugs to alter performance in the cognitive domain of executive function. Five adult female rhesus monkeys (ages of 9-17years old) were trained on the object retrieval with detours (ORD) task of executive function. For the ORD task, the monkeys were required to retrieve food items from a clear box with one open end that was rotated to different positions along with varying placements of food. When the non-selective BZ triazolam and the α1GABAA-preferring agonists zolpidem and zaleplon were evaluated in the ORD task, deficits in performance occurred at doses that did not increase the latency of monkeys to initiate responding and/or increase the percentage of reaches that were incorrect (i.e., reaches in which food was not obtained). Cognition-impairing effects of triazolam and zolpidem in ORD were blocked by the α1GABAA-preferring antagonist, ßCCT, whereas the α5GABAA-preferring antagonist XLi-093 blocked the effects of triazolam but not zolpidem. While these findings suggest a role for both α1GABAA and α5GABAA receptor mechanisms, α1GABAA receptor mechanisms appear to be sufficient for impairments in executive function induced by BZ-type drugs.
Asunto(s)
Benzodiazepinas/efectos adversos , Cognición/efectos de los fármacos , Función Ejecutiva/efectos de los fármacos , Macaca mulatta/fisiología , Macaca mulatta/psicología , Receptores de GABA-A/clasificación , Receptores de GABA-A/efectos de los fármacos , Acetamidas/efectos adversos , Animales , Ansiolíticos/efectos adversos , Benzodiazepinonas/farmacología , Carbolinas/farmacología , Cognición/fisiología , Función Ejecutiva/fisiología , Femenino , Antagonistas de Receptores de GABA-A/farmacología , Hipnóticos y Sedantes/efectos adversos , Imidazoles/farmacología , Piridinas/efectos adversos , Pirimidinas/efectos adversos , Receptores de GABA-A/fisiología , Triazolam/efectos adversos , ZolpidemRESUMEN
Genetic and pharmacological studies have demonstrated that α2- and α4-containing GABA(A) receptors mediate the anxiolytic effects of a number of agents. Flavonoids are a class of ligands that act at GABA(A) receptors and possess anxiolytic effects in vivo. Here we demonstrate that the synthetic flavonoid, 3-hydroxy-2'-methoxy-6-methylflavone (3-OH-2'MeO6MF) potentiates GABA-induced currents at recombinant α1/2ß2, α1/2/4/6ß1-3γ2L but not α3/5ß1-3γ2L receptors expressed in Xenopus oocytes. The enhancement was evident at micromolar concentrations (EC(50) values between 38 and 106 µM) and occurred in a flumazenil-insensitive manner. 3-OH-2'MeO6MF displayed preference for ß2/3- over ß1-containing receptors with the highest efficacy observed at α2ß2/3γ2L, displaying a 4-11-fold increase in efficacy over α2ß1γ2L and α1/4/6-containing subtypes. In contrast, 3-OH-2'MeO6MF acted as a potent bicuculline-sensitive activator, devoid of potentiation effects at extrasynaptic α4ß2/3δ receptors expressed in oocytes. The affinity of 3-OH-2'MeO6MF for α4ß2/3δ receptors (EC(50) values between 1.4 and 2.5 µM) was 10-fold higher than at α4ß1δ GABA(A) receptors. 3-OH-2'MeO6MF acted as a full agonist at α4ß2/3δ (105% of the maximal GABA response) but as a partial agonist at α4ß1δ (61% of the maximum GABA response) receptors. In mice, 3-OH-2'MeO6MF (1-100 mg/kg i.p.) induced anxiolytic-like effects in two unconditioned models of anxiety: the elevated plus maze and light/dark paradigms. No sedative or myorelaxant effects were detected using holeboard, actimeter and horizontal wire tests and only weak barbiturate potentiating effects on the loss of righting reflex test. Taken together, these data suggest that 3-OH-2'MeO6MF is an anxiolytic without sedative and myorelaxant effects acting through positive allosteric modulation of the α2ß2/3γ2L and direct activation of α4ß2/3δ GABA(A) receptor subtypes.
Asunto(s)
Ansiolíticos/farmacología , Flavonas/farmacología , Receptores de GABA-A/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Ansiolíticos/química , Diazepam/farmacología , Relación Dosis-Respuesta a Droga , Ácidos Grasos Volátiles , Flavonas/administración & dosificación , Flavonas/efectos adversos , Flavonas/química , Flumazenil/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Actividad Motora/efectos de los fármacos , Oocitos , Pentilenotetrazol/farmacología , Receptores de GABA-A/clasificación , Receptores de GABA-A/efectos de los fármacos , Proteínas Recombinantes , Tiopental/administración & dosificación , Tiopental/farmacología , Xenopus , Ácido gamma-Aminobutírico/química , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
GABA(A) (γ-aminobutyric acid, type A) receptors are a family of ligand-gated ion channels that are essential for the regulation of central nervous system function. Benzodiazepines - which non-selectively target GABA(A) receptors containing the α1, α2, α3 or α5 subunits - have been in clinical use for decades and are still among the most widely prescribed drugs for the treatment of insomnia and anxiety disorders. However, their use is limited by side effects and the risk of drug dependence. In the past decade, the identification of separable key functions of GABA(A) receptor subtypes suggests that receptor subtype-selective compounds could overcome the limitations of classical benzodiazepines; furthermore, they might be valuable for novel indications such as chronic pain, depression, schizophrenia, cognitive enhancement and stroke.
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Benzodiazepinas/farmacología , Moduladores del GABA/uso terapéutico , Receptores de GABA-A/efectos de los fármacos , Analgésicos/farmacología , Animales , Trastornos de Ansiedad/tratamiento farmacológico , Trastorno Depresivo/tratamiento farmacológico , Moduladores del GABA/farmacología , Humanos , Hipnóticos y Sedantes/farmacología , Mutación/genética , Mutación/fisiología , Nootrópicos/farmacología , Receptores de GABA-A/clasificación , Receptores de GABA-A/genética , Esquizofrenia/tratamiento farmacológico , Trastornos del Inicio y del Mantenimiento del Sueño/tratamiento farmacológico , Accidente Cerebrovascular/tratamiento farmacológico , Trastornos Relacionados con SustanciasRESUMEN
Recent GABA(A) receptor drug discovery efforts have culminated in the development of transgenic mice and subtype-selective pharmacological tools, enhancing our understanding of the major inhibitory neural system in the central nervous system. Notably, subtype-selective tools have demonstrated in both preclinical studies and, to some extent, in man that it is possible to develop drugs that share the clinical benefits of benzodiazepines (e.g., anxiolysis) while obviating some adverse effects of these clinically important drugs. Here, we highlight chronic pain as another therapeutic area in which subtype-selective GABA(A) receptor drugs might have clinical utility. Specifically, based on research in animal models of inflammatory/neuropathic pain, we suggest that subtype-selective positive modulators of GABA(A) alpha(2/3) receptors might reverse a loss of postsynaptic GABA(A) receptor-mediated inhibitory function in spinal cord, leading to analgesia. However, alteration of presynaptic inhibitory neural transmission in chronic pain suggests that drugs that negatively modulate GABA(A) receptors might also be effective analgesics. For example, the non-selective negative allosteric modulator FG-7142 reverses allodynia in an animal model of neuropathic pain. Importantly, these two mechanisms are not mutually exclusive. Further clinical exploration in pain of available positive and negative subtype-selective modulators that have been administered to humans would considerably aid back translation, allowing for improved therapeutic development.
Asunto(s)
Analgésicos/farmacología , Moduladores del GABA/farmacología , Receptores de GABA-A/efectos de los fármacos , Regulación Alostérica , Animales , Descubrimiento de Drogas , Humanos , Dolor/tratamiento farmacológico , Dolor/fisiopatología , Receptores de GABA-A/clasificación , Receptores de GABA-A/fisiologíaRESUMEN
6-Hydroxyflavone (6HF), a naturally occurring flavonoid, was previously reported to bind to type A gamma-aminobutyric acid (GABA(A)) receptors benzodiazepine (BZ) site with moderate binding affinity. In the present study, we showed that 6HF partially potentiated GABA-induced currents in native GABA(A) receptors expressed in cortical neurons via BZ site, as the enhancement was blocked by the antagonist flumazenil. Furthermore, in patch clamp studies, 6HF displayed significant preference for alpha(2)- and alpha(3)-containing subtypes, which were thought to mediate anxiolytic effect, compared to alpha(1)- and alpha(5)-containing subtypes expressed in HEK 293T cells. In mice, 6HF exhibited anxiolytic-like effect in the elevated plus-maze test, unaccompanied at anxiolytic doses by the sedative, cognitive impairing, myorelaxant, motor incoordination and anticonvulsant effects commonly associated with classical BZs when tested in the hole-board, step-through passive avoidance, horizontal wire, rotarod, and pentylenetetrazol (PTZ)-induced seizure tests, respectively. The findings therefore identified 6HF as a promising drug candidate for the treatment of anxiety-like disorders.
Asunto(s)
Ansiolíticos/farmacología , Flavonoides/farmacología , Receptores de GABA-A/clasificación , Animales , Ataxia/inducido químicamente , Células Cultivadas , Flunitrazepam/química , Flunitrazepam/farmacología , Humanos , Masculino , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Estructura Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , RatasRESUMEN
Nonselective benzodiazepines exert their pharmacological effects via GABAA receptors containing either an alpha1, alpha2, alpha3, or alpha5 subunit. The use of subtype-selective tool compounds along with transgenic mice has formed the conceptual framework for defining the requirements of subtype-selective compounds with potentially novel pharmacological profiles. More specifically, compounds which allosterically modulate the alpha2 and/or alpha3 subtypes but are devoid of, or have much reduced, effects at the alpha1 subtype are hypothesized to be anxioselective (i.e., anxiolytic but devoid of sedation). Accordingly, three compounds, MRK-409, TPA023 and TPA023B, which selectively potentiated the effects of GABA at the alpha2 and alpha3 compared to alpha1 subtypes were progressed into man. All three compounds behaved as nonsedating anxiolytics in preclinical (rodent and primate) species but, surprisingly, MRK-409 produced sedation in man at relatively low levels of occupancy (< 10%). This sedation liability of MRK-409 in man was attributed to its weak partial agonist efficacy at the alpha1 subtype since both TPA023 and TPA023B lacked any alpha1 efficacy and did not produce overt sedation even at relatively high levels of occupancy (> 50%). The anxiolytic efficacy of TPA023 was evaluated in Generalized Anxiety Disorder and although these clinical trials were terminated early due to preclinical toxicity issues, the combined data from these incomplete studies demonstrated an anxiolytic-like effect of TPA023. This compound also showed a trend to increase cognitive performance in a small group of schizophrenic subjects and is currently under further evaluation of its cognition-enhancing effects in schizophrenia as part of the TURNS initiative. In contrast, the fate of the back-up clinical candidate TPA023B has not been publicly disclosed. At the very least, these data indicate that the pharmacological profile of compounds that differentially modulate specific populations of GABAA receptors is distinct from classical benzodiazepines and should encourage further preclinical and clinical investigation of such compounds, with the caveat that, as exemplified by MRK-409, the preclinical profile might not necessarily translate into man.
Asunto(s)
Ansiolíticos/uso terapéutico , Trastornos de Ansiedad/tratamiento farmacológico , Benzodiazepinas/uso terapéutico , Agonistas de Receptores de GABA-A/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Hidrocarburos Fluorados/farmacología , Piridazinas/farmacología , Triazoles/farmacología , Animales , Ansiolíticos/efectos adversos , Benzodiazepinas/efectos adversos , Humanos , Receptores de GABA-A/clasificación , Resultado del TratamientoRESUMEN
The gamma-aminobutyric acid (GABA)(A) receptors mediating the discriminative stimulus effects of ethanol were studied by comparing the potency of ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazol(1,5-a)benzodiazepine-3-carboxylate (Ro15-4513) and ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol(1,5-a)-benzodiazepine-3-carboxylate (flumazenil, Ro15-1788) to antagonize ethanol, pentobarbital (PB), and midazolam substitution for ethanol. Ro15-4513 has high affinity for receptors containing alpha(4/6) and alpha(5) subunits and lower affinity for alpha(1), alpha(2), and alpha(3) subunits. Flumazenil is nonselective for GABA(A) receptors containing alpha(1), alpha(2), alpha(3), and alpha(5) subunits and has low affinity for alpha(4/6)-containing receptors. Male (n = 9) and female (n = 8) cynomolgus monkeys (Macaca fascicularis) were trained to discriminate ethanol (1.0 or 2.0 g/kg i.g., 30-min pretreatment) from water. Ethanol, PB, and midazolam dose-dependently substituted for ethanol (80% ethanol-appropriate responding). Ro15-4513 (0.003-0.56 mg/kg i.m., 5-min pretreatment) shifted the ethanol, PB, and midazolam dose-response functions rightward in a vast majority of monkeys tested (15/15, 16/17, and 11/12, respectively). In contrast, flumazenil (0.30-10.0 mg/kg i.m., 5-min pretreatment) shifted the ethanol, PB, and midazolam dose-response functions rightward in 9 of 16, 12 of 16, and 7 of 9 monkeys tested, respectively. In the monkeys showing antagonism with both Ro15-4513 and flumazenil, ethanol and PB substitution were antagonized more potently by Ro15-4513 than by flumazenil, whereas midazolam substitution was antagonized with similar potency. There were no sex or training dose differences, with the exception that flumazenil failed to antagonize ethanol substitution in males trained to discriminate 2.0 g/kg ethanol. GABA(A) receptors with high affinity for Ro15-4513 (i.e., containing alpha(4/6) and alpha(5) subunits) may be particularly important mediators of the multiple discriminative stimulus effects of ethanol through GABA(A) receptor systems.
Asunto(s)
Aprendizaje Discriminativo/fisiología , Etanol/antagonistas & inhibidores , Midazolam/antagonistas & inhibidores , Pentobarbital/antagonistas & inhibidores , Subunidades de Proteína/fisiología , Receptores de GABA-A/fisiología , Animales , Azidas/farmacología , Benzodiazepinas/farmacología , Aprendizaje Discriminativo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Etanol/farmacología , Femenino , Flumazenil/farmacología , Macaca fascicularis , Masculino , Midazolam/farmacología , Pentobarbital/farmacología , Subunidades de Proteína/clasificación , Receptores de GABA-A/clasificaciónRESUMEN
This mini-review attempts to update experimental evidence on the existence of GABA(A) receptor pharmacological subtypes and to produce a list of those native receptors that exist. GABA(A) receptors are chloride channels that mediate inhibitory neurotransmission. They are members of the Cys-loop pentameric ligand-gated ion channel (LGIC) superfamily and share structural and functional homology with other members of that family. They are assembled from a family of 19 homologous subunit gene products and form numerous receptor subtypes with properties that depend upon subunit composition, mostly hetero-oligomeric. These vary in their regulation and developmental expression, and importantly, in brain regional, cellular, and subcellular localization, and thus their role in brain circuits and behaviors. We propose several criteria for including a receptor hetero-oligomeric subtype candidate on a list of native subtypes, and a working GABA(A) receptor list. These criteria can be applied to all the members of the LGIC superfamily. The list is divided into three categories of native receptor subtypes: "Identified", "Existence with High Probability", and "Tentative", and currently includes 26 members, but will undoubtedly grow, with future information. This list was first presented by Olsen & Sieghart (in press).
Asunto(s)
GABAérgicos/farmacología , Activación del Canal Iónico/efectos de los fármacos , Receptores de GABA-A/clasificación , Receptores de GABA-A/fisiología , Animales , Canales de Cloruro/efectos de los fármacos , Inhibición Neural/efectos de los fármacosRESUMEN
In this review we attempt to summarize experimental evidence on the existence of defined native GABA(A) receptor subtypes and to produce a list of receptors that actually seem to exist according to current knowledge. This will serve to update the most recent classification of GABA(A) receptors (Pharmacol Rev 50:291-313, 1998) approved by the Nomenclature Committee of the International Union of Pharmacology. GABA(A) receptors are chloride channels that mediate the major form of fast inhibitory neurotransmission in the central nervous system. They are members of the Cys-loop pentameric ligand-gated ion channel (LGIC) superfamily and share structural and functional homology with other members of that family. GABA(A) receptors are assembled from a family of 19 homologous subunit gene products and form numerous, mostly hetero-oligomeric, pentamers. Such receptor subtypes with properties that depend on subunit composition vary in topography and ontogeny, in cellular and subcellular localization, in their role in brain circuits and behaviors, in their mechanisms of regulation, and in their pharmacology. We propose several criteria, which can be applied to all the members of the LGIC superfamily, for including a receptor subtype on a list of native hetero-oligomeric subtypes. With these criteria, we develop a working GABA(A) receptor list, which currently includes 26 members, but will undoubtedly be modified and grow as information expands. The list is divided into three categories of native receptor subtypes: "identified," "existence with high probability," and "tentative."
Asunto(s)
Subunidades de Proteína/clasificación , Receptores de GABA-A/clasificación , Terminología como Asunto , Animales , Sistema Nervioso Central/metabolismo , Humanos , Ligandos , Conformación Proteica , Subunidades de Proteína/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismoRESUMEN
It is known that gastric mechanoreceptor stimuli are widely integrated into neuronal circuits that involve visceral nuclei of hindbrain as well as several central brain areas. GABAergic neurons are widely represented in hindbrain nuclei controlling gastric motor functions, but limited information is available specifically about GABA(A)-responding neurons in brain visceral areas. The present investigation was designed to determine the central sensory neuronal pathways and their GABA(A)-alpha1 and -alpha3 receptor presenting neurons that respond to gastric mechanoreceptor stimulation within the entire rat brain. Low pressure gastric distension was used to deliver physiological mechanical stimuli in anesthetized rats, and different protocols of gastric distension were performed to mimic different stimulation patterns with and without sectioning vagal and/or splanchnic afferent nerves. Mapping of activated neurons was investigated using double colorimetric immunohistochemistry for GABA(A)-alpha1 or -alpha3 subunits and c-Fos. Following stomach distension, neurons expressing GABA(A) receptors with alpha1 or alpha3 subunits were detected. Low frequency gastric distension induced c-Fos expression in nucleus tractus solitarii (NTS) only, whereas in the high frequency gastric distension c-Fos positive nuclei were found in lateral reticular nucleus and in NTS in addition to some forebrain areas. In contrast, during the tonic-rapid gastric distension the neuronal activation was found in hindbrain, midbrain and forebrain areas. Moreover different protocols of gastric stimulation activated diverse patterns of neurons presenting GABA(A)-alpha1 or -alpha3 receptors within responding brain nuclei, which may indicate a probable functional significance of differential expression of GABA(A)-responding neurons. The same protocol of gastric distension performed in vagotomized rats has confirmed the primary role of the vagus in the response of activation of gastric brain areas, whereas neuronal input of splanchnic origins was shown to play an important role in modulating the mechanogastric response of brain areas.
Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica/fisiología , Receptores de GABA-A/metabolismo , Estómago/inervación , Aferentes Viscerales/fisiología , Animales , Encéfalo/citología , Recuento de Células , Masculino , Estimulación Física/métodos , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/clasificación , Aferentes Viscerales/citologíaRESUMEN
AIM: To clone and analyse the coding cDNA sequence of alpha1, beta2 and gamma2 subunit of GABAA receptor in American king Pigeon. METHODS: Withdrew total RNA from the American king pigeon brain, reverse transcribing general primers to acquire a gene set cDNA. Designing specific primers of three subunit mRNA of the GABAA receptor, by RT-PCR respectively expanded the conservative gene of al subunit, beta2 subunit and gamma2 subunit of GABAA receptor, and carried on clone, plastid identification and the sequence measurese of three genes. RESULTS: The experiment on sequence measures has succeeded that sequence analysis indicated that lengths of the conservative gene of alpha1 subunit, beta2 subunit and gamma2 subunit of GABAA receptor was respectively 899 bp, 597 bp and 563 bp, homology on reference sequence was respectively 94.99%, 94.64% and 96.28%. CONCLUSION: Homology is high on the conservative gene of alpha1 subunit, beta2 subunit and gamma2 subunit of GABAA receptor of brain tissue of pigeon and chicken but there is a discriminating characteristic in different kinds of animals.