RESUMEN
High-affinity transporters for norepinephrine (NE) and serotonin (5-HT), which ensure neurotransmitter clearance at the synapse, are the principal targets of widely used antidepressant drugs. Antidepressants targeting these high-affinity transporters, however, do not provide positive treatment outcomes for all patients. Other monoamine transport systems, with lower affinity, have been detected in the brain, but their role is largely unknown. Here we report that OCT2, a member of the polyspecific organic cation transporter (OCT) family, is expressed notably in the limbic system and implicated in anxiety and depression-related behaviors in the mouse. Genetic deletion of OCT2 in mice produced a significant reduction in brain tissue concentrations of NE and 5-HT and in ex vivo uptake of both these neurotransmitters in the presence of the dual 5-HT-NE transport blocker, venlafaxine. In vivo clearance of NE and 5-HT evaluated using microiontophoretic electrophysiology was diminished in the hippocampus of OCT2(-/-) mice in the presence of venlafaxine, thereby affecting postsynaptic neuronal activity. OCT2(-/-) mice displayed an altered sensitivity to acute treatments with NE- and/or 5-HT-selective transport blockers in the forced-swim test. Moreover, the mutant mice were insensitive to long-term venlafaxine treatment in a more realistic, corticosterone-induced, chronic depression model. Our findings identify OCT2 as an important postsynaptic determinant of aminergic tonus and mood-related behaviors and a potential pharmacological target for mood disorders therapy.
Asunto(s)
Antidepresivos de Segunda Generación/uso terapéutico , Encéfalo/metabolismo , Ciclohexanoles/uso terapéutico , Depresión/tratamiento farmacológico , Norepinefrina/metabolismo , Proteínas de Transporte de Catión Orgánico/fisiología , Serotonina/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Antidepresivos de Segunda Generación/farmacología , Ansiedad/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Corticosterona , Ciclohexanoles/farmacología , Depresión/inducido químicamente , Modelos Animales de Enfermedad , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiología , Sistema Límbico/efectos de los fármacos , Sistema Límbico/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Imagen Molecular/métodos , Imagen Molecular/psicología , Proteínas de Transporte de Catión Orgánico/biosíntesis , Proteínas de Transporte de Catión Orgánico/genética , Transportador 2 de Cátion Orgánico , Ensayo de Unión Radioligante/métodos , Ensayo de Unión Radioligante/psicología , Clorhidrato de VenlafaxinaRESUMEN
By taking up serotonin (5-hydroxytryptamine, 5-HT) released in the extracellular space, the 5-HT transporter (5-HTT) regulates central 5-HT neurotransmission. Possible adaptive changes in 5-HT neurotransmission in knock-out mice that do not express the 5-HT transporter were investigated with special focus on 5-HT1A and 5-HT1B receptors. Specific labelling with radioligands and antibodies, and competitive RT-PCR, showed that 5-HT1A receptor protein and mRNA levels were significantly decreased in the dorsal raphe nucleus (DRN), increased in the hippocampus and unchanged in other forebrain areas of 5-HTT-/- vs. 5-HTT+/+ mice. Such regional differences also concerned 5-HT1B receptors because a decrease in their density was found in the substantia nigra (-30%) but not the globus pallidus of mutant mice. Intermediate changes were noted in 5-HTT+/- mice compared with 5-HTT+/+ and 5-HTT-/- animals. Quantification of [35S]GTP-gamma-S binding evoked by potent 5-HT1 receptor agonists confirmed such changes as a decrease in this parameter was noted in the DRN (-66%) and the substantia nigra (-30%) but not other brain areas in 5-HTT-/- vs. 5-HTT+/+ mice. As expected from actions mediated by functional 5-HT1A and 5-HT1B autoreceptors, a decrease in brain 5-HT turnover rate after i.p. administration of ipsapirone (a 5-HT1A agonist), and an increased 5-HT outflow in the substantia nigra upon local application of GR 127935 (a 5-HT1B/1D antagonist) were observed in 5-HTT+/+ mice. Such effects were not detected in 5-HTT-/- mice, further confirming the occurrence of marked alterations of 5-HT1A and 5-HT1B autoreceptors in these animals.
Asunto(s)
Química Encefálica/genética , Proteínas Portadoras/genética , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Receptores de Serotonina/genética , Animales , Autorreceptores/genética , Autorreceptores/metabolismo , Unión Competitiva/fisiología , Proteínas Portadoras/metabolismo , Citalopram/farmacología , Dipéptidos/metabolismo , Dipéptidos/farmacología , Regulación hacia Abajo/genética , Proteínas de Unión al GTP/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Ácido Hidroxiindolacético/análisis , Ácido Hidroxiindolacético/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxadiazoles/farmacología , Piperazinas/metabolismo , Piperazinas/farmacología , Piridinas/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , ARN Mensajero/análisis , Receptor de Serotonina 5-HT1B , Receptores de Serotonina/metabolismo , Receptores de Serotonina 5-HT1 , Serotonina/análisis , Serotonina/metabolismo , Antagonistas de la Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Agonistas de Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Compuestos de Espiro/metabolismo , Compuestos de Espiro/farmacología , Sustancia Negra/metabolismo , Radioisótopos de Azufre , Transmisión Sináptica/fisiología , TritioRESUMEN
With the aim of exploring the relationship between the serotonin transporter (5-HTT or SERT) and the activity level of serotonin (5-HT) neurotransmission, in vivo expression of this protein was specifically altered using a nonviral DNA transfer method. Plasmids containing the entire coding sequence or a partial antisense sequence of the 5-HTT gene were complexed with the cationic polymer polyethylenimine and injected into the dorsal raphe nucleus of adult male rats. Significant increase or decrease in both [(3)H]citalopram binding and [(3)H]5-HT synaptosomal uptake were observed in various brain areas up to 2 weeks after a single administration of the sense plasmid or 7 d after injection of the short antisense plasmid, respectively. Such changes in 5-HTT expression were associated with functional alterations in 5-HT neurotransmission, as shown by the increased capacity of 5-HT(1A) receptor stimulation to enhance [(35)S]GTP-gamma-S binding onto the dorsal raphe nucleus in sections from rats injected with the sense plasmid. Conversely, both a decrease in 5-HT(1A)-mediated [(35)S]GTP-gamma-S binding and a reduced potency of the 5-HT(1A) receptor agonist ipsapirone to inhibit neuronal firing were observed in the dorsal raphe nucleus of antisense plasmid-injected rats. Furthermore, changes in brain 5-HT and/or 5-HIAA levels, and sleep wakefulness circadian rhythm in the latter animals demonstrated that altered expression of 5-HTT by recombinant plasmids has important functional consequences on central 5-HT neurotransmission in adult rats.
Asunto(s)
Proteínas Portadoras/fisiología , Corteza Cerebral/fisiología , Técnicas de Transferencia de Gen , Glicoproteínas de Membrana/fisiología , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Neuronas/fisiología , Núcleos del Rafe/fisiología , Serotonina/metabolismo , Transmisión Sináptica/fisiología , Animales , Autorradiografía , Proteínas Portadoras/genética , Línea Celular , Citalopram/farmacocinética , ADN sin Sentido , Dopamina/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Homeostasis , Masculino , Glicoproteínas de Membrana/genética , Neuronas/efectos de los fármacos , Plásmidos , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Serotonina/fisiología , Receptores de Serotonina 5-HT1 , Proteínas Recombinantes/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Agonistas de Receptores de Serotonina/farmacología , Porcinos , Transmisión Sináptica/efectos de los fármacos , TransfecciónRESUMEN
The serotonin transporter (5-HTT) plays a key-role in the control of serotoninergic neurotransmission and is the target of some antidepressants. Possible adaptive changes in brain 5-HT2A receptors were investigated in knock-out mice that do not express the 5-HTT. Autoradiographic labeling of these receptors by the selective antagonist [3H]MDL 100,907 and saturation experiments with cortical membranes revealed: (1) a new localization of these receptors in the external field of striatum (possibly in striosomes); (2) regional variations in adaptive changes in the density of 5-HT2A receptors in 5-HT(-/-) mutants (-30-40% in the claustrum, cerebral cortex and lateral striatum; no significant change in the striatum core) as compared to wild-type mice.
Asunto(s)
Proteínas Portadoras/fisiología , Corteza Cerebral/metabolismo , Glicoproteínas de Membrana/fisiología , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Receptores de Serotonina/metabolismo , Adaptación Fisiológica , Animales , Sitios de Unión , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Corteza Cerebral/citología , Regulación hacia Abajo , Fluorobencenos/metabolismo , Heterocigoto , Homocigoto , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Piperidinas/metabolismo , Receptor de Serotonina 5-HT2A , Antagonistas de la Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana PlasmáticaRESUMEN
-The increased delivery of serotonin (5-hydroxytryptamine, 5-HT) to the lung aggravates the development of hypoxia-induced pulmonary hypertension in rats, possibly through stimulation of the proliferation of pulmonary artery smooth muscle cells (PA-SMCs). In cultured rat PA-SMCs, 5-HT (10(-8) to 10(-6) mol/L) induced DNA synthesis and potentiated the mitogenic effect of platelet-derived growth factor-BB (10 ng/mL). This effect was dependent on the 5-HT transporter (5-HTT), since it was prevented by the 5-HTT inhibitors fluoxetine (10(-6) mol/L) and paroxetine (10(-7) mol/L), but it was unaltered by ketanserin (10(-6) mol/L), a 5-HT2A receptor antagonist. In PA-SMCs exposed to hypoxia, the levels of 5-HTT mRNA (measured by competitive reverse transcriptase-polymerase chain reaction) increased by 240% within 2 hours, followed by a 3-fold increase in the uptake of [3H]5-HT at 24 hours. Cotransfection of the cells with a construct of human 5-HTT promoter-luciferase gene reporter and of pCMV-beta-galactosidase gene allowed the demonstration that exposure of cells to hypoxia produced a 5.5-fold increase in luciferase activity, with no change in beta-galactosidase activity. The increased expression of 5-HTT in hypoxic cells was associated with a greater mitogenic response to 5-HT (10(-8) to 10(-6) mol/L) in the absence as well as in the presence of platelet-derived growth factor-BB. 5-HTT expression assessed by quantitative reverse transcriptase-polymerase chain reaction and in situ hybridization in the lungs was found to predominate in the media of pulmonary artery, in which a marked increase was noted in rats that had been exposed to hypoxia for 15 days. These data show that in vitro and in vivo exposure to hypoxia induces, via a transcriptional mechanism, 5-HTT expression in PA-SMCs, and that this effect contributes to the stimulatory action of 5-HT on PA-SMC proliferation. In vivo expression of 5-HTT by PA-SMC may play a key role in serotonin-mediated pulmonary vascular remodeling.
Asunto(s)
Proteínas Portadoras/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Proteínas de Transporte de Membrana , Mitógenos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Proteínas del Tejido Nervioso , Arteria Pulmonar/efectos de los fármacos , Serotonina/farmacología , Animales , Proteínas Portadoras/genética , División Celular , Hipoxia de la Célula , Hipertensión Pulmonar/etiología , Pulmón/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Regiones Promotoras Genéticas , Arteria Pulmonar/citología , Arteria Pulmonar/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Wistar , Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Timidina/metabolismoRESUMEN
The functional role of the dopamine transporter (DAT) in central dopaminergic neurotransmission was assessed further by investigating the consequences on dopamine (DA) turn-over of up- and down-regulation of this protein induced by a non-viral gene transfer approach. For this purpose, expression plasmids containing the sense or antisense coding sequence of DAT complexed with the cationic polymer, polyethylenimine (PEI), were injected into the rat substantia nigra, the brain region containing the majority of DA cell bodies. Before in vivo injection, the efficacies of the different DNA constructs were assessed by transfection studies into LLC-PK1 cells. Stereotaxic administration of the sense plasmid complexed to PEI induced, 3 days later, a significant increase in the immunoautoradiographic labelling by anti-DAT antibodies of the substantia nigra and various DA projection areas. These effects were associated with a significantly enhanced capacity of striatal synaptosomes to take up [3H]-DA and lasted up to 14 days postinjection. In contrast, 7 days after intranigral administration of the antisense plasmid complexed to PEI, we observed a significant decrease of DAT immunolabelling in the substantia nigra and [3H]-DA uptake by striatal synaptosomes. Whereas DA turnover in the striatum was unaltered 3 days after intranigral administration of the sense plasmid, it was increased 7 days after intranigral administration of the antisense construct. These data indicate that non-viral transfer of the sense or antisense coding sequence of DAT can be used as a novel approach to induce long-term changes in central DA neurotransmission.
Asunto(s)
Química Encefálica/genética , Proteínas Portadoras/genética , Regulación hacia Abajo/fisiología , Técnicas de Transferencia de Gen , Glicoproteínas de Membrana , Proteínas de Transporte de Membrana , Regulación hacia Arriba/fisiología , Ácido 3,4-Dihidroxifenilacético/análisis , Factores de Edad , Animales , Aniones/química , Elementos sin Sentido (Genética)/farmacología , Proteínas Portadoras/metabolismo , ADN Recombinante/química , ADN Recombinante/farmacología , Dopamina/análisis , Dopamina/metabolismo , Dopamina/farmacocinética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Expresión Génica , Células LLC-PK1/fisiología , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Plásmidos/farmacología , Ratas , Ratas Sprague-Dawley , Serotonina/análisis , Serotonina/metabolismo , Sustancia Negra , Porcinos , Transfección , TritioRESUMEN
In order to map the recently cloned serotonin 5-HT6 receptor in the rat brain and spinal cord, polyclonal antibodies were raised against a synthetic octadecapeptide corresponding to a specific portion (Leu398-Val415) of the C-terminal domain of this receptor. Antibodies were detected by enzyme-linked immunosorbent assay as soon as one month after the first injection to rabbits of the peptide coupled to keyhole limpet hemocyanin. Immunoautoradiographic experiments with antibodies affinity-purified on Affi-Gel coupled to the peptide antigen showed that 5-HT6-like immunoreactive material was abundant in the olfactory tubercle (plexiform layer), cerebral cortex (frontal and entorhinal areas), nucleus accumbens, striatum, hippocampus (strata oriens and radiatum of the CA1 area, molecular layer of the dentate gyrus) and the molecular layer of the cerebellum. A specific immunolabeling, but at moderate intensity, was also observed in the thalamus, substantia nigra, superficial layer of the superior colliculus, motor trigeminal nucleus and facial nucleus. In contrast, no 5-HT6-like immunoreactive material was found in white matter areas. As the regional distribution of 5-HT6 receptor-like immunoreactivity matched generally that previously found for the 5-HT6 receptor mRNA, one could infer that this receptor protein is addressed in the vicinity of its synthesis site, i.e. on somas and/or dendrites. Indeed, immunohistochemistry at the light and electron microscope level showed that 5-HT6-like immunoreactivity was associated with dendritic processes in both the striatum and the dentate gyrus of the hippocampus. The relative abundance of 5-HT6 receptor-like immunoreactivity in extrapyramidal and limbic areas suggests that 5-HT6 receptors may participate in the serotoninergic control of motor function and mood-dependent behavior, respectively.
Asunto(s)
Sistema Nervioso Central/química , Receptores de Serotonina/análisis , Receptores de Serotonina/genética , Animales , Especificidad de Anticuerpos , Autorradiografía , Núcleo Caudado/química , Núcleo Caudado/citología , Sistema Nervioso Central/citología , Ensayo de Inmunoadsorción Enzimática , Hipocampo/química , Hipocampo/citología , Inmunohistoquímica , Masculino , Microscopía Electrónica , Datos de Secuencia Molecular , Neuronas/química , Neuronas/ultraestructura , Neuropéptidos/análisis , Neuropéptidos/inmunología , Putamen/química , Putamen/citología , Conejos , Ratas , Ratas Sprague-Dawley , Receptores de Serotonina/inmunología , Homología de Secuencia de AminoácidoRESUMEN
Possible adaptive changes of the recently cloned serotonin 5-HT6 receptor after the selective lesion of serotoninergic neurons by an intracerebral administration of 5,7-dihydroxytryptamine were investigated using competitive RT-PCR (reverse transcription followed by polymerase chain reaction) for the measurement of 5-HT6-mRNA in various areas of the rat central nervous system. In control rats, 5-HT6-mRNA was the most abundant in the nucleus accumbens, followed by the olfactory tubercle and the striatum. High levels of 5-HT6-mRNA were also found in the hypothalamus and the hippocampus, whereas the cerebral cortex, the substantia nigra, and the spinal cord contained moderate levels of the transcript. Low but easily quantifiable levels of 5-HT6-mRNA were measured in the ventral tegmental area, the anterior raphe area, and the cerebellum. In addition, moderate to low levels of this mRNA were also found in dorsal root ganglia and the pituitary gland. Three weeks after the microinfusion of 5,7-dihydroxytryptamine into the anteroventral vicinity of the dorsal raphe nucleus in nomifensine-pretreated rats, the levels of serotonin transporter-mRNA were reduced by 90% in the anterior raphe area, as expected of the extensive lesion of serotoninergic neurons. In contrast, quantitative determinations of the 5-HT6-mRNA in this area as well as in the nucleus accumbens, the striatum, and the hippocampus indicated that its levels were not significantly different in 5,7-dihydroxytryptamine-treated rats and in controls. These data showed that the 5-HT6 receptor: 1) is not an autoreceptor, and 2) exhibits probably no up regulation in postsynaptic target cells after the selective degeneration of serotoninergic projections.
Asunto(s)
5,7-Dihidroxitriptamina/farmacología , Sistema Nervioso Central/metabolismo , ARN Mensajero/metabolismo , Receptores de Serotonina/biosíntesis , Serotoninérgicos/farmacología , Animales , Sistema Nervioso Central/efectos de los fármacos , Hibridación in Situ , Masculino , Oligonucleótidos Antisentido/farmacología , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Receptores de Serotonina/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
DNA fragments from a genomic library were used to establish the partial structure of the human dopamine D3 receptor gene (DRD3). Its coding sequence contains 6 exons and stretches over 40,000 base pairs. The complete DRD3 transcript and three shorter variants, in which the second and/or third exon are deleted, were detected in similar proportions in brains from four controls and three psychiatric patients. The Msp I polymorphism was localized in the fifth intron of the gene, 40,000 base pairs downstream the Bal I polymorphism and a PCR-based method was developed for genotyping this polymorphism. The distribution of the Msp I and Bal I genotypes were not independent in 297 individuals (chi 2 = 10.5, df = 4, P = 0.03), but only a weak association was found between allele 1 of the Bal I polymorphism and allele 2 of the Msp I polymorphism (chi 2 = 3.99, df = 1, P = 0.04). The previously reported association between homozygosity at both alleles of the Bal I polymorphism and schizophrenia was presently maintained in an extended sample, comprising 119 DSM-III-R chronic schizophrenics and 85 controls (chi 2 = 5.3, df = 1, P = 0.02) and found more important in mal than in females. The presence of the Bal I allele 2 is associated with an early age at onset, particularly in males (df = 35, t value = 2.6, P = 0.014). In the same sample, allelic frequencies, genotype counts, and proportion of homozygotes for the Msp I polymorphism did not differ between schizophrenics and controls (chi 2 = 0.06, df = 1, P = 0.80, chi 2 = 0.22, df = 1, P = 0.90 and chi 2 = 0.16, df = 1, P = 0.69, respectively). The large distance of the Msp I polymorphism from the Bal I polymorphism and its localization in the 3' part of the gene may explain the discrepant results obtained with the two polymorphisms.
Asunto(s)
Receptores de Dopamina D2/genética , Esquizofrenia/genética , Animales , Secuencia de Bases , Cartilla de ADN , Desoxirribonucleasa HpaII , Desoxirribonucleasas de Localización Especificada Tipo II , Genotipo , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , ARN Mensajero/genética , Ratas , Receptores de Dopamina D3RESUMEN
The recent demonstration of the occurrence in rat brain and other nonpancreatic tissues of carboxypeptidase A (CPA) gene transcripts without associated catalytic activity could be ascribed to the presence of a soluble endogenous protein inhibitor. This tissue carboxypeptidase inhibitor (TCI), detected by the inhibition of added bovine pancreatic CPA, was purified from rat brain. Peptides were obtained by partial proteolysis of purified TCI, a protein of approximately 30 kDa, and starting from their sequences, a full-length cDNA encoding a 223-amino acid protein containing three potential phosphorylation sites was cloned from a cDNA library. Its identity with TCI was shown by expression in Escherichia coli of a recombinant protein recognized by antibodies raised against native TCI and display characteristic CPA-inhibiting activity. TCI appears as a hardly reversible, non-competitive, and potent inhibitor of CPA1 and CPA2 (Ki approximately 3 nM) and mast-cell CPA (Ki = 16 nM) and inactive on various other proteases. This pattern of selectivity might be attributable to a limited homology of a 11-amino acid sequence with sequences within the activation segments of CPA and CPB known to interact with residues within their active sites. The widespread expression of TCI in a number of tissues (e.g., brain, lung, or digestive tract) and its apparently cytosolic localization point to a rather general functional role, e.g., in the control of cytosolic protein degradation.
Asunto(s)
Carboxipeptidasas/antagonistas & inhibidores , Glicoproteínas/biosíntesis , Glicoproteínas/aislamiento & purificación , Páncreas/metabolismo , Inhibidores de Proteasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Antígenos , Secuencia de Bases , Encéfalo/metabolismo , Bovinos , Clonación Molecular , ADN Complementario , Expresión Génica , Glicoproteínas/farmacología , Cinética , Mamíferos , Mastocitos/metabolismo , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacología , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Porcinos , Transcripción GenéticaRESUMEN
Numerous studies on the involvement of dopamine receptors in the genetics of alcoholism focused on associations between a polymorphism of the D2 dopamine receptor (DRD2) gene and alcohol dependence. However, the results of these studies are conflicting. Another receptor, the D3 dopamine receptor (DRD3), may be of additional interest since it is specifically located in the limbic area, and in particular in the nucleus accumbens which plays a significant role in the reward process of addiction behavior. We thus tested the association in three independent samples of alcoholic patients, with different origins and various inclusion criteria. No difference in the DRD3 gene polymorphism emerged between controls and alcoholic patients, regardless of their origin, inclusion criteria, or presence or absence of the DRD2 TaqI A1-allele. Despite the fact that more information could have been considered and that association studies provide limited information, there is good evidence that this DRD3 polymorphism does not play a major role in the genetic component of alcoholism.
Asunto(s)
Alcoholismo/genética , Receptores de Dopamina D2/genética , ADN/genética , Femenino , Humanos , Masculino , Receptores de Dopamina D3RESUMEN
The serotonin 5-HT3-A receptor (5-HT3R-A) mRNA has been shown recently to be expressed as two forms (5-HT3R-AL and 5-HT3R-AS) varying by the presence or the absence of a sequence of 18 bases in the region corresponding to the second cytoplasmic domain of the receptor, and generated by alternative splicing at the level of the 3' acceptor site of exon 9. As the long form of the receptor exhibits a potential phosphorylation site that is disrupted by the alternative splicing, the hypothesis of functional identity and stochastic expression of these two variants was questioned. In the present study, we used quantitative reverse transcriptase-polymerase chain reaction to examine the possible influence of culture conditions on the expression and the alternative splicing of 5-HT3R-A mRNA in NG108-15 clonal cells. Cell differentiation induced by dibutyryl cyclic AMP or theophyllin plus prostaglandin E1 in the presence of 10% serum reduced by threefold the expression of total 5-HT3R-A mRNA, and favored the short form of the message as the ratio S/L (5-HT3R-AS mRNA/5-HT3R-AL mRNA) shifted from 2.23 to 7.33 after 9 days of treatment. Culture with 0.3% serum (instead of 10%) lowered by 10-fold the level of expression of total 5-HT3R-A mRNA, but only slightly reduced the S/L ratio. However, this ratio fell to 0.06 in the presence of 0.3% serum plus 10 ng/ml basic fibroblast growth factor. These results demonstrate that external factors can influence the differential expression of the two variants of the 5-HT3R-A in NG108-15 cells. Appropriate culture conditions for the almost exclusive expression of 5-HT3R-AS mRNA or 5-HT3R-AL mRNA in NG108-15 cells should allow the identification of possible differences in the respective functional properties of each of these two forms of the native 5-HT3 receptor.
Asunto(s)
Empalme Alternativo , ARN Mensajero/metabolismo , Receptores de Serotonina/genética , Animales , Secuencia de Bases , Adhesión Celular , Diferenciación Celular , Línea Celular , Medio de Cultivo Libre de Suero , Dimetilsulfóxido/farmacología , Sustancias de Crecimiento/farmacología , Células Híbridas/citología , Células Híbridas/fisiología , Ratones , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/genética , RatasRESUMEN
The functions of the D4 receptor, a newly cloned D2-like receptor, as well as the identity of cells expressing it, are still poorly defined. Using quantitative polymerase chain reaction we detected the messenger RNA of the D4, but not other D2-like receptor, in cultured granule cells from neonatal rat cerebellum. In these neurons, dopamine reduced high-voltage-activated calcium current, with a pharmacology corresponding to that of the D4 receptor. The response declined from one to three days, when calcium currents were mostly sensitive to nifedipine, to 15 days, when nifedipine-insensitive calcium currents were also present and D4 receptor messenger RNA had declined. The dopamine response was abolished after pretreatment of the cells by pertussis toxin, was potentiated and made irreversible by infusion of guanosine 5'-O-(3-thiotriphosphate) but persisted in the presence of cyclic AMP and isobutylmethylxanthine. These results indicate the presence in the neonatal cerebellum of a functional D4 receptor inhibiting an L-type calcium current, an action involving a Gi/Go protein but independent from adenylate cyclase inhibition.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Cerebelo/metabolismo , Agonistas de Dopamina/farmacología , Receptores de Dopamina D2 , Receptores Dopaminérgicos/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Animales Recién Nacidos , Secuencia de Bases , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Electrofisiología , Inmunohistoquímica , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , ARN Complementario/biosíntesis , Ratas , Ratas Wistar , Receptores Dopaminérgicos/efectos de los fármacos , Receptores de Dopamina D4 , Transducción de Señal/efectos de los fármacosRESUMEN
The expression of central 5-HT1A and 5-HT1B receptors was studied in several brain areas of rats subjected to a 2-week period of chronic alcoholization, followed by 18 h withdrawal. Quantitative autoradiography indicated that the ethanol treatment provoked an increase (approximately +30%) in the labeling by [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) and [3H]N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide ([3H]WAY-100635) of 5-HT1A autoreceptors in the dorsal raphe nucleus, accompanied by a concomitant decrease in the labeling of postsynaptic 5-HT1A receptors in the hippocampus (approximately -20%), anterior (approximately -30%) and posterior (approximately -32%) cortices. These changes were associated with a tendency toward an increase and decrease in 5-HT1A mRNA levels in the anterior raphe area and hippocampus, respectively, suggesting that the changes observed are due to modifications in 5-HT1A receptor protein synthesis. The autoradiographic labeling of 5-HT1B receptors by serotonin-O-carboxymethylglycyl[125I]iodotyrosinamide ([125I]GTI) was found to increase (+55%) in the globus pallidus of alcoholized rats. Interestingly, a significant increase (+57%) in 5-HT1B receptor mRNA levels was observed in the striatum, which contains cell bodies of neurons projecting into the globus pallidus. These data suggest that altered sensitivity of chronically alcoholized rats to 5-HT1A and 5-HT1B receptor ligands may result from alcohol-induced changes in the transcription of the genes encoding these receptors.
Asunto(s)
Encéfalo/efectos de los fármacos , Etanol/farmacología , Receptores de Serotonina/efectos de los fármacos , Animales , Autorradiografía , Expresión Génica , Masculino , ARN Mensajero/biosíntesis , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Factores de TiempoAsunto(s)
Antipsicóticos/farmacología , Encéfalo/fisiología , Agonistas de Dopamina/farmacología , Receptores Dopaminérgicos/efectos de los fármacos , Animales , Bradiquinina/farmacología , Carbacol/farmacología , División Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Dopamina/metabolismo , Dopamina/farmacología , Agonistas de Dopamina/metabolismo , Ergolinas/farmacología , Expresión Génica/efectos de los fármacos , Genes fos , Haloperidol/farmacología , Humanos , Fosfatos de Inositol/metabolismo , Neurotensina/metabolismo , Oxidopamina/farmacología , Quinpirol , Receptores Dopaminérgicos/clasificación , Sistemas de Mensajero Secundario , Transducción de Señal , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
We have established the cellular distribution of the dopamine D3 receptor using tritiated 7-hydroxy-N-N-di-n-propyl-2-aminotetralin and a complementary RNA probe to visualize autoradiographically the protein in binding studies and the gene transcripts by in situ hybridization, respectively. Studies with these two markers confirm the restricted expression of the D3 receptor in few brain areas, i.e. mainly the ventral striatal complex, the substantia nigra-ventral tegmental area and the cerebellum. In nucleus accumbens, the D3 receptor was mainly expressed in medium-sized neurons of the rostral pole and ventromedial shell subdivisions, but not of the core or septal pole, i.e. accumbal subdivisions expressing the D2 receptor. In the ventromedial shell, about 60% of the D3 receptor-expressing neurons were neurotensin neurons, presumably projecting to the ventral pallidum. In the islands of Calleja, both D3 receptor binding and messenger RNA were abundant in the entire population of granule cells. These cells are known to make sparse contacts with dopaminergic axons and also to express the D1 receptor. In the mesencephalon, low levels of D3 messenger RNA were detected in few dopamine neurons of substantia nigra pars lateralis and ventral tegmental area. In addition, some D3 receptor binding but not messenger RNA was detected in medial substantia nigra and lateral ventral tegmental area, where the receptor is presumably located presynaptically on afferents. In the archicerebellum, Purkinje cell perikarya in lobules 9 and 10 expressed the D3 receptor messenger RNA, whereas binding sites were found in the molecular layer, where corresponding dendrites but no known dopaminergic projection from mesencephalon are found. The occurrence of D3 receptor gene expression in some brain areas receiving low dopamine innervation supports the hypothesis that this receptor may mediate non-synaptic actions of dopamine.
Asunto(s)
Neuronas/fisiología , Núcleo Accumbens/fisiología , Receptores Dopaminérgicos/genética , Animales , Autorradiografía , Sitios de Unión , Expresión Génica , Hibridación in Situ , Masculino , ARN Mensajero/genética , Ratas , Ratas Wistar , Sustancia Negra/fisiología , Tetrahidronaftalenos , Tirosina 3-Monooxigenasa/genéticaRESUMEN
The effects of interruption of dopaminergic transmission or sustained blockade of dopamine receptors by neuroleptics on the dopamine D3 receptor in the shell of the nucleus accumbens were investigated in rats. In this brain area the D3 receptor is abundant and may mediate antipsychotic drug effects. The D3 receptor density and mRNA abundance were evaluated with 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin and by quantitative PCR or image analysis of in situ hybridization signals, respectively. Unilateral dopamine neuron degeneration by 6-hydroxydopamine or sections triggered, after a few days, a marked decrease (up to 50%) in D3 receptor binding and mRNA in the nucleus accumbens. In contrast, a 2-week treatment with the neuroleptic haloperidol (20 mg/kg) had no effect on D3 receptor density and mRNA but enhanced D2 receptor density and mRNA level by > 50%. In addition, tolerance to the haloperidol-induced change of neurotensin mRNA mediated by the D2 receptor developed, but there was no tolerance to the opposite change mediated by the D3 receptor. Reserpine, a monoamine-depleting drug with antipsychotic activity, did not modify D3 receptor mRNA. These observations reinforce the idea that the D3 receptor may be an important target for neuroleptics whose antipsychotic actions, but not extrapyramidal motor actions, do not display tolerance. The D3 receptor mRNA level was also decreased by a unilateral injection in dopamine cell body areas of colchicine, a drug blocking the anterograde axonal transport, or by baclofen, a type A gamma-aminobutyric acid receptor agonist reducing dopamine neuron activity, but not by sustained blockade of D1-like and D2-like, neurotensin, or cholecystokinin receptors. We therefore propose that an anterograde factor present in mesolimbic dopaminergic neurons, but distinct from dopamine and known peptide cotransmitters, plays a positive role on transcription of the D3 receptor gene.
Asunto(s)
Encéfalo/metabolismo , Dopamina/fisiología , Neuronas/fisiología , Receptores de Dopamina D2 , Receptores Dopaminérgicos/metabolismo , Animales , Mapeo Encefálico , Antagonistas de Dopamina/farmacología , Expresión Génica/efectos de los fármacos , Hibridación in Situ , Masculino , Núcleo Accumbens/metabolismo , Oxidopamina/farmacología , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores Dopaminérgicos/genética , Receptores de Dopamina D3 , Reserpina/farmacologíaRESUMEN
Using in situ hybridization histochemistry in rat nucleus accumbens, we show that the dopamine D3 receptor mRNA is expressed in the ventromedial part of the shell subdivision, where its gross distribution matches that of neurotensin mRNA. In addition, hybridization studies at the cellular level show that a large fraction of the neurotensin neurons co-express the D3 receptor mRNA in this restricted area. In contrast, the dopamine D2 receptor mRNA is expressed mainly in the core and marginally in the shell, at the level of the cone. In rats treated by haloperidol and sulpiride, two D2-like receptor antagonists, but not by SCH 23390, a D1-like receptor antagonist, proneurotensin mRNA was increased in the D2 receptor mRNA-rich areas but decreased in the D3 receptor mRNA-rich areas. This suggests that the D2 and D3 receptors control neurotensin mRNA expression negatively and positively, respectively.
Asunto(s)
Neurotensina/biosíntesis , Núcleo Accumbens/metabolismo , ARN Mensajero/biosíntesis , Receptores de Dopamina D2/fisiología , Receptores Dopaminérgicos/fisiología , Animales , Benzazepinas/farmacología , Haloperidol/farmacología , Masculino , Ratas , Ratas Wistar , Receptores de Dopamina D3 , Sulpirida/farmacología , Transcripción GenéticaRESUMEN
Transfection of a human dopamine D3 receptor cDNA in a neuroblastoma-glioma hybrid cell line (NG 108-15) provided clonal cell lines stably expressing up to 600 fmol per mg protein of [125I]iodosulpiride binding sites. Dopamine and several agonists distinguished two receptor-affinity states in membranes. In the case of dopamine, the high-affinity state (Ki = 0.9 nM, 30% of total binding) was completely converted into a low-affinity state (Ki = 57 nM) in the presence of 10 microM guanosine-5'-O-(3-thiotriphosphate). In addition to these two sites, a site with a very low affinity for dopamine was evidenced in whole cells. The dopamine D3 receptor mediated two responses: c-fos activation, as measured by the appearance of Fos-like immunoreactivity, and increased mitogenesis, as measured by incorporation of [3H]thymidine. The Fos-like immunoreactivity appeared within 30 min, lasted 2 h and was blocked by the partially selective dopamine D3 receptor compound (+)-UH 232 (cis-(+)-5-methoxy-1-methyl-2-(di-n-propylamino)tetralin). The mitogenic effect, which occurred after a lag time (over 2 h stimulation), was produced with subnanomolar potency and full intrinsic activity by several compounds previously identified as dopamine D2 receptor agonists, e.g. quinpirole, (+)-7-OH-DPAT ((+)-7-hydroxy-2-(di-n-propylamino)tetralin) and RU 24926 (N-n-propyl-di-beta(3-hydroxyphenyl)-ethylamine), and was reversibly blocked by (+)-UH 232 (Ki = 9 nM). Talipexole (B-HT 920, 5-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin) was identified as a partial agonist at the dopamine D3 receptor. Dopamine D3 receptor-mediated mitogenesis was potentiated by a phorbol ester and was abolished by pretreatment with pertussis toxin. A mitogenic effect of same amplitude was elicited by bradykinin or carbachol, both acting through constitutive receptors. Bradykinin markedly activated inositol phosphate turnover, and had no effect on forskolin-stimulated cyclic AMP accumulation. Carbachol inhibited forskolin-stimulated cyclic AMP accumulation and had no effect on inositol-phosphate turnover. Quinpirole had no effect on any of these second messenger pathways. Thus, in transfected NG 108-15 cells, the dopamine D3 receptor is coupled to a pertussis toxin-sensitive G protein and mediates two possibly unrelated biological effects, through initial biochemical events that remain to be identified.
Asunto(s)
Receptores de Dopamina D2 , Receptores Dopaminérgicos/fisiología , Animales , AMP Cíclico/metabolismo , Ergolinas/farmacología , Proteínas de Unión al GTP/fisiología , Genes fos , Glioma/patología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Células Híbridas , Ratones , Neuroblastoma/patología , Quinpirol , Ratas , Receptores de Dopamina D3 , Sulpirida/metabolismo , Timidina/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Our knowledge of dopamine receptor diversity has markedly increased during the past few years as a result of discovery of five distinct genes, splice variants and polymorphic receptors. The genes can be classified in two subfamilies: the intronless genes that encode the D1 and D5 receptors positively linked to adenylyl cyclase and genes with introns that encode the two isoforms of the D2 receptor and the D3 and D4 receptors. The various dopamine receptor subtypes can be distinguished by their sequence, intracellular signalling systems, pharmacology and localisation. The localisation of the D3 receptor in the shell of nucleus accumbens suggests its participation in brain reward circuits and actions of substances of abuse.