RESUMEN
BACKGROUND: The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor in the immunoglobulin superfamily. RAGE is localized throughout ascending sensory pathways (skin, peripheral nerve, dorsal root ganglion, spinal cord), and in cell types interacting with sensory neurons (endothelial cells, smooth muscle cells, monocytes and macrophages). Neuronal RAGE expression increases in pathological pain states in humans and rodents, and soluble RAGE attenuates thermal hypoalgesia in diabetic mice. The objective of the present study was to investigate whether pharmacological modulation of RAGE could attenuate mechanical allodynia in rodent pain models. METHODS: We developed an anti-RAGE monoclonal antibody (11E6) that binds to the C2 immunoglobulin domain of human RAGE, binds to mouse RAGE, and presumably to the same domain in mouse RAGE. The antinociceptive activity of 11E6 was investigated in mouse models of inflammatory (complete Freund's adjuvant) and neuropathic (chronic constriction injury of the sciatic nerve) pain. Mice were dosed intraperitoneally with 11E6 or IgG (negative control). RESULTS: Increased mechanical thresholds were observed following a single dose of 11E6 in both inflammatory and neuropathic pain models. Similar treatment with IgG did not alter nociceptive sensitivity. Repeated dosing with 11E6 significantly attenuated established mechanical hypersensitivity in a neuropathic pain model in a dose-related fashion. CONCLUSIONS: These data demonstrate that specific modulation of RAGE effectively attenuates nociceptive sensitivity associated with chronic inflammatory and neuropathic pain states.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Factores Inmunológicos/uso terapéutico , Neuralgia/tratamiento farmacológico , Receptor para Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Animales , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Modelos Animales de Enfermedad , Femenino , Adyuvante de Freund , Ganglios Espinales/metabolismo , Humanos , Hiperalgesia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuralgia/etiología , Neuronas Aferentes/metabolismo , Receptor para Productos Finales de Glicación Avanzada/inmunología , Nervio Ciático/metabolismo , Médula Espinal/metabolismoRESUMEN
BACKGROUND AND PURPOSE: The histamine H4 receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In this report, we describe the in vitro and in vivo anti-inflammatory properties of a potent histamine H4 receptor antagonist, A-940894 (4-piperazin-1-yl-6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-d]pyrimidin-2-ylamine). EXPERIMENTAL APPROACH: We have analysed the pharmacological profile of A-940894 at mouse native, rat recombinant and human recombinant and native, histamine H4 receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan-induced peritonitis. KEY RESULTS: A-940894 potently binds to both human and rat histamine H4 receptors and exhibits considerably lower affinity for the human histamine H1, H2 or H3 receptors. It potently blocked histamine-evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine-induced shape change of mouse bone marrow-derived mast cells and chemotaxis of human eosinophils in vitro. In a mouse mast cell-dependent model of zymosan-induced peritonitis, A-940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D2 levels. Finally, A-940894 has good pharmacokinetic properties, including half-life and oral bioavailability in rats and mice. CONCLUSIONS AND IMPLICATIONS: These data suggest that A-940894 is a potent and selective histamine H4 receptor antagonist with pharmacokinetic properties suitable for long-term in vivo testing and could serve as a useful tool for the further characterization of histamine H4 receptor pharmacology.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Piperazinas/farmacología , Pirimidinas/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/farmacocinética , Unión Competitiva , Calcio/metabolismo , Forma de la Célula , Quimiotaxis , Eosinófilos/efectos de los fármacos , Eosinófilos/fisiología , Femenino , Histamina/farmacología , Humanos , Masculino , Mastocitos/citología , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Peritonitis/inducido químicamente , Peritonitis/tratamiento farmacológico , Peritonitis/inmunología , Piperazinas/farmacocinética , Prostaglandina D2/metabolismo , Pirimidinas/farmacocinética , ARN Mensajero/biosíntesis , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/genética , Receptores Histamínicos/biosíntesis , Receptores Histamínicos/genética , Receptores Histamínicos H4 , Proteínas Recombinantes/antagonistas & inhibidores , ZimosanRESUMEN
The histamine H3 receptor, first described in 1983 as a histamine autoreceptor and later shown to also function as a heteroreceptor that regulates the release of other neurotransmitters, has been the focus of research by numerous laboratories as it represents an attractive drug target for a number of indications including cognition. The purpose of this review is to acquaint the reader with the current understanding of H3 receptor localization and function as a modulator of neurotransmitter release and its effects on cognitive processes, as well as to provide an update on selected H3 antagonists in various states of preclinical and clinical advancement. Blockade of centrally localized H3 receptors by selective H3 receptor antagonists has been shown to enhance the release of neurotransmitters such as histamine, ACh, dopamine and norepinephrine, among others, which play important roles in cognitive processes. The cognitive-enhancing effects of H3 antagonists across multiple cognitive domains in a wide number of preclinical cognition models also bolster confidence in this therapeutic approach for the treatment of attention deficit hyperactivity disorder, Alzheimer's disease and schizophrenia. However, although a number of clinical studies examining the efficacy of H3 receptor antagonists for a variety of cognitive disorders are currently underway, no clinical proof of concept for an H3 receptor antagonist has been reported to date. The discovery of effective H3 antagonists as therapeutic agents for the novel treatment of cognitive disorders will only be accomplished through continued research efforts that further our insights into the functions of the H3 receptor.
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Trastornos del Conocimiento/tratamiento farmacológico , Receptores Histamínicos H3/efectos de los fármacos , Receptores Histamínicos H3/fisiología , Animales , Cognición/fisiología , Histamina/fisiología , Antagonistas de los Receptores Histamínicos H3/farmacología , Antagonistas de los Receptores Histamínicos H3/uso terapéutico , Humanos , Memoria/efectos de los fármacos , Neurotransmisores/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
The past decade has been marked by a dramatic increase in the availability of techniques to identify and clone genes that are differentially expressed in disease states and by drug treatments. The applications of such techniques to problems in biological psychiatry are manifold and the implications of discovering novel and/or known genes that are perturbed in neuropsychiatric disorders profound. While there are success stories, it is becoming ever more apparent that each of these techniques has its limitations, particularly when applied to the central nervous system. Given that these methods (e.g. differential display, RNA fingerprinting, suppression-subtractive hybridization, microarrays) are labour-intensive and potentially time-consuming, it is important to understand these limitations. For example, differential display is capable of detecting very small changes in the expression of mRNA species. Methods like suppression-subtractive hybridization are better suited to examine potential differences in rare transcripts, but only when their expression is changed substantially (currently ? 5-fold). Moreover, both the functional and morphological organization of the central nervous system present challenges that may not be encountered in other systems. In this overview, we will discuss the advantages and disadvantages of some of these approaches and their application to research in biological psychiatry.
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Psiquiatría Biológica/métodos , Perfilación de la Expresión Génica/métodos , Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Técnica del ADN Polimorfo Amplificado Aleatorio/métodos , Animales , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , HumanosRESUMEN
Imidazobenzodiazepines such as RY-80 have been reported to exhibit both high affinity and selectivity for GABA(A) receptors containing an alpha(5) subunit. A single amino acid residue (alpha(5)Ile215) has been identified that plays a critical role in the high-affinity, subtype-selective effects of RY-80 and structurally related ligands. Thus, substitution of alpha(5)Ile215 with the cognate amino acid contained in the alpha(1) subunit (Val211) reduced the selectivity of RY-80 for alpha(5)beta(3)gamma(2) receptors from approximately 135- to approximately 8-fold compared with alpha(1)beta(3)gamma(2) receptors. This mutation produced a comparable reduction in the selectivity of RY-24 (a structural analog of RY-80) for alpha(5)beta(3)gamma(2) receptors but did not markedly alter the affinities of ligands (e.g., flunitrazepam) that are not subtype-selective. Conversely, substitution of the alpha(1) subunit with the cognate amino acid contained in the alpha(5) subunit (i.e., alpha(1)V211I) increased the affinities of alpha(5)-selective ligands by a approximately 20-fold and reduced by 3-fold the affinity of an alpha(1)-selective agonist (zolpidem). Increasing the lipophilicity (e.g., by substitution of Phe) of alpha(5)215 did not significantly affect the affinities (and selectivities) of RY-80 and RY-24 for alpha(5)-containing GABA(A) receptors. However, the effect of introducing hydrophilic and or charged residues (e.g., Lys, Asp, Thr) at this position was no greater than that produced by the alpha(5)I215V mutation. These data indicate that residue alpha(5)215 may not participate in formation of the lipophilic L(2) pocket that has been proposed to contribute to the unique pharmacological properties of alpha(5)-containing GABA(A) receptors. RY-80 and RY-24 acted as inverse agonists in both wild-type alpha(5)beta(3)gamma(2) and mutant alpha(5)I215Kbeta(3)gamma(2) receptors expressed in Xenopus laevis oocytes. However, both RY-24 and RY-80 acted as antagonists at mutant alpha(5)I215Vbeta(3)gamma(2) and alpha(5)I215Tbeta(3)gamma(2) receptors, whereas the efficacy of flunitrazepam was similar at all three receptor isoforms. The data demonstrate that amino acid residue alpha(5)215 is a determinant of both ligand affinity and efficacy at GABA(A) receptors containing an alpha(5) subunit.
Asunto(s)
Isoleucina/metabolismo , Receptores de GABA-A/metabolismo , Alquinos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Benzodiazepinas/farmacología , Relación Dosis-Respuesta a Droga , Imidazoles/farmacología , Isoleucina/genética , Ligandos , Datos de Secuencia Molecular , Mutagénesis , Oocitos , Ratas , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/genética , Homología de Secuencia de Aminoácido , Transfección , Tritio , Xenopus laevisRESUMEN
Studies using recombinant systems indicate that 'diazepam-insensitive' GABA(A) receptors in the central nervous system contain alpha4 and alpha6 subunits while 'diazepam-sensitive' GABA(A) receptors contain alpha1, alpha2, alpha3 and alpha5 subunits. Both native and recombinant diazepam-sensitive GABA(A) receptors typically exhibit large, outwardly rectifying currents. For example, in patch clamp studies, Human Embryonic Kidney (HEK) 293 cells transfected with cDNAs encoding alpha1beta2gamma2 subunits exhibit a rectification ratio (I +60 mV/I -60 mV) of 1.95 +/- 0.21. However, anomalous rectification was observed in recombinant diazepam-insensitive GABA(A) receptors composed of either alpha4beta2gamma2 (rectification ratio, 0.74 +/- 0.09) or alpha6beta2gamma2 (rectification ratio, 0.67 +/- 0.11) subunits. Based on sequence differences between diazepam-sensitive and -insensitive GABA(A) receptor alpha subunits in the vicinity of the putative channel lining, a point mutation was introduced at His273 on the alpha4 subunit. The rectification ratio in cells expressing a mutated alpha4(Asn273)beta2gamma2 receptor increased to 1.92 +/- 0.17. Moreover, mutation of the homologous residue in the alpha1 subunit to histidine reduced the rectification ratio of alpha1(His274)beta2gamma2 to 1.02 +/- 0.12. The affinities of benzodiazepine site ligands at diazepam-sensitive and -insensitive GABA(A) receptors were unaffected by these mutations. Thus, the electrophysiological properties of diazepam-sensitive and -insensitive GABA(A) receptors may be as divergent as their pharmacological characteristics.
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Diazepam/farmacología , Moduladores del GABA/farmacología , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/genética , Secuencia de Aminoácidos , Línea Celular , Electrofisiología , Humanos , Isomerismo , Ligandos , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Mutación Puntual/fisiología , Ensayo de Unión RadioliganteRESUMEN
Most general anesthetics produce two distinct actions at GABA(A) receptors. Thus, these drugs augment GABA-gated chloride currents (referred to as an indirect action) and, at higher concentrations, elicit chloride currents in the absence of GABA (referred to as a direct action). Because a beta subunit appears to be required for the direct action of intravenous anesthetics in recombinant GABA(A) receptors, site-directed mutagenesis of the beta3 subunit was performed to identify amino acid residues that are critical for this action. In HEK293 cells expressing a prototypical GABA(A) receptor composed of alpha1beta3gamma2 subunits, mutation of amino acid 290 from Asn to Ser dramatically reduced both etomidate-induced chloride currents and its ability to stimulate [3H]flunitrazepam binding. By contrast, the ability of etomidate to augment GABA-gated chloride currents and GABA-enhanced [3H]flunitrazepam binding was retained. The demonstration that the direct, but not the indirect, actions of etomidate are dependent on beta3(Asn290) indicates that the dual actions of this intravenous anesthetic at GABA(A) receptors are mediated via distinct loci.
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Anestésicos Intravenosos/farmacología , Etomidato/farmacología , Receptores de GABA-A/fisiología , Animales , Línea Celular , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/fisiología , Flunitrazepam/metabolismo , Humanos , Sustancias Macromoleculares , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Mutación Puntual , Ratas , Receptores de GABA-A/biosíntesis , Receptores de GABA-A/efectos de los fármacos , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Antidepressant therapies include drugs with a remarkable structural diversity and non-pharmacological interventions, such as electroconvulsive shock. Although the primary neurochemical effects of these treatments may differ, the > or = 2- to 3-wk lag in therapeutic onset has led to the hypothesis that adaptive changes in a final common pathway are required for an antidepressant action. Based on this hypothesis, we sought to identify and characterize common changes in gene expression following chronic antidepressant treatments. We utilized a differential display strategy to identify genes that were differentially expressed in mice following chronic treatment with imipramine and electroconvulsive shock. Differential display PCR followed by subcloning, screening by reverse Northern blot, and confirmation by Northern blot revealed a significant increase in the expression of one gene candidate from mouse cortex following antidepressant treatments. The sequence of this 193-bp gene candidate was an exact match to the DNA sequence of mouse mitochondrial cytochrome b. In contrast to the increased mRNA levels of cytochrome b found in cortex, chronic treatment with these antidepressants did not alter mRNA levels in hippocampus, cerebellum, or liver. Moreover, no differences in cortical levels of cytochrome b mRNA were observed after either acute antidepressant treatments or chronic treatment with nonantidepressant drugs (haloperidol and morphine). The observation that chronic, but not acute treatment with imipramine and electroconvulsive shock produces a region-specific change in the levels of mRNA encoding cytochrome b suggests that this enzyme may be involved in the sequence of events resulting in an antidepressant action.
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Antidepresivos/administración & dosificación , Corteza Cerebral/efectos de los fármacos , Grupo Citocromo b/genética , ARN Mensajero/efectos de los fármacos , Animales , Corteza Cerebral/metabolismo , Grupo Citocromo b/efectos de los fármacos , Grupo Citocromo b/metabolismo , Esquema de Medicación , Electrochoque/efectos adversos , Regulación de la Expresión Génica/efectos de los fármacos , Haloperidol/administración & dosificación , Imipramina/administración & dosificación , Inyecciones Intraperitoneales , Masculino , Ratones , Morfina/administración & dosificación , ARN Mensajero/metabolismoRESUMEN
The cDNA, containing the complete human alpha-1-antitrypsin (AT) sequence starting from codon 2, was used to construct bacterial strains producing AT. The fusion protein was obtained by junction of the AT cDNA to the fragment of an Escherichia coli ompF gene. We have also modified the AT cDNA's 5'-terminal part to construct DNAs containing ATG-codon and cDNA sequences starting from codons 1 or 2. These DNAs were inserted into E. coli expression vectors pBR322/trpII-8 and pKK223-3 that allow transcription from efficient trp- and tac-promoters. This construction resulted in the induction of a 46 kDa protein. The polypeptide produced was recognized by an antiserum raised against human alpha-1-antitrypsin protein. Truncation of the gene at its 5'-end or synthesis as a fusion OmpF-AT protein increases expression up to 10-fold, to a level of approximately 1%. On the contrary, no dependence on the promoter type has been observed. Physical properties of the recombinant proteins are discussed.