RESUMEN

RATIONALE: The serotonin (5-HT) system is considered important for decision-making. However, its role in reward- and punishment-based processing has not yet been clearly determined. OBJECTIVES: The present study examines the effect of 5-HTTLPR genotype and tryptophan depletion on reward- and punishment-related processing, using a task that considers decision-making in situations of subtlety of choice. Thus, it considers that response choice often occurs in situations where both options are desirable (e.g., choosing between mousse au chocolat or crème caramel cheesecake from a menu) or undesirable. It also considers that response choice is easier when the reinforcements associated with the options are far apart, rather than close, in value. MATERIALS AND METHODS: Healthy volunteers underwent acute tryptophan depletion (ATD) or control procedures and genotyping of the 5-HTTLPR for long and short allele variants. We then examined the effects and interactions of ATD and the serotonin promoter polymorphism genotype on two aspects of decision-making: (a) decision form, choosing between two objects to gain the greater reward or lesser punishment and (b) between-object reinforcement distance, the difference in reinforcements associated with two options. RESULTS: ATD and LL homozygosity had comparable interactions with decision form and between-object reinforcement distance. Specifically, both modulated the effect of between-object reinforcement distance when deciding between objects both associated with punishment. Moreover, ATD and genotype interacted with ATD disproportionately affecting the performance of the LL homozygous group. CONCLUSIONS: These results suggest that serotonin is particularly associated with punishment, rather than reward-related processing, and that individual sensitivity to punishment-related information and tryptophan depletion varies with genotype.

Asunto(s)

Conducta de Elección/fisiología , Genotipo , Polimorfismo Genético/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Triptófano/fisiología , Adulto , Alelos , Aminoácidos/administración & dosificación , Cápsulas , Toma de Decisiones/fisiología , Método Doble Ciego , Femenino , Juego de Azar/psicología , Tamización de Portadores Genéticos , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Motivación , Reconocimiento Visual de Modelos , Regiones Promotoras Genéticas/genética , Castigo , Refuerzo en Psicología , Triptófano/administración & dosificación

RESUMEN

The neuropeptide galanin (GAL) is widely expressed in the central nervous system. Animal studies have implicated GAL in alcohol abuse and anxiety: chronic ethanol intake increases hypothalamic GAL mRNA; high levels of stress increase GAL release in the central amygdala. The coding sequence of the galanin gene, GAL, is highly conserved and a functional polymorphism has not yet been found. The aim of our study was, for the first time, to identify GAL haplotypes and investigate associations with alcoholism and anxiety. Seven single-nucleotide polymorphisms (SNPs) spanning GAL were genotyped in 65 controls from five populations: US and Finnish Caucasians, African Americans, Plains and Southwestern Indians. A single haplotype block with little evidence of historical recombination was observed for each population. Four tag SNPs were then genotyped in DSM-III-R lifetime alcoholics and nonalcoholics from two population isolates: 514 Finnish Caucasian men and 331 Plains Indian men and women. Tridimensional Personality Questionnaire harm avoidance (HA) scores, a dimensional measure of anxiety, were obtained. There was a haplotype association with alcoholism in both the Finnish (P=0.001) and Plains Indian (P=0.004) men. The SNPs were also significantly associated. Alcoholics were divided into high and low HA groups (>or= and

Asunto(s)

Alcoholismo/genética , Población Negra/genética , Etnicidad/genética , Galanina/genética , Indígenas Norteamericanos/genética , Población Blanca/genética , Ansiedad/genética , Secuencia de Bases , Cartilla de ADN , Femenino , Finlandia , Genotipo , Reducción del Daño , Humanos , Masculino , Encuestas y Cuestionarios , Estados Unidos

RESUMEN

A significant proportion of the human genome is contained within haplotype blocks across which pairwise linkage disequilibrium (LD) is very high. However, LD is also often high between markers at more remote distances, and within different haplotype blocks. Here, we evaluate the origins of haplotype block structure in the three genes for alpha1 adrenergic receptors (alpha1-AR) in the human genome ( ADRA1A, ADRA1B and ADRA1D) by genotyping dense single-nucleotide polymorphism (SNP) marker maps, and show that LD signals between distant markers are due to the presence of extended haplotype superblocks in individuals with ancient chromosomes which have escaped historic recombination. ARs mediate the physiological effects of epinephrine and norepinephrine, and are targets of many therapeutic drugs. This work has identified haplotype backgrounds of alpha1-AR missense variants, haplotype block structures in US Caucasians and African Americans, and haplotype tag SNPs for each block, and we present strong evidence for ancient haplotype block superstructure at these genes which has been partially disrupted by recombination, and evidence for reinstatement of linkage disequilibrium by subsequent recombination events. ADRA1A is comprised of four haplotype blocks in US Caucasians, while in African Americans Block 1 is split. ADRA1B has four blocks in US Caucasians, but in African Americans only the first two blocks are present. ADRA1D has two blocks in US Caucasians, and the first block is replaced by two smaller blocks in African Americans. For both ADRA1A and ADRA1B, haplotype superstructures may represent a novel, higher-level hierarchy in the human genome, which may reduce redundancy of testing by further aggregation of genotype data.

Asunto(s)

Genoma Humano , Haplotipos/genética , Desequilibrio de Ligamiento , Receptores Adrenérgicos alfa 1/genética , Negro o Afroamericano , Genotipo , Humanos , Polimorfismo de Nucleótido Simple , Estados Unidos , Población Blanca

RESUMEN

The major inflammatory cytokines interleukin(IL)1beta, IL6 and tumor necrosis factor alpha (TNFalpha) play a crucial role in infection, inflammation and stress responses. Previously, three coding genes were resequenced, identifying promoter polymorphisms that were used in association studies of neurodegenerative diseases, metabolic disorders and cancer. These studies have produced intriguing but inconsistent results, potentially because the known functional variants: IL1B-511 C>T, IL6-174 G>C and TNF-308 G>A provided an incomplete picture of the total functional diversity at these genes. Therefore, we created marker panels for IL1B, IL6 and TNF/LTA that included the known functional marker but also other markers evenly spaced and with sufficient density to identify haplotype block structure and to maximize haplotype diversity. A total of 26 markers were genotyped in 96 US Caucasians and 96 African Americans. In both populations, a single block with little evidence of historical recombination was observed in IL1B, IL6 and TNF/LTA. For each gene, haplotypes captured the information content of each functional locus, even if that locus was not genotyped, and presumably haplotypes would capture the signal from unknown functional loci whose alleles are of moderate abundance. This study demonstrates the utility of using gene haplotype maps and marker panels as tools for linkage studies on related phenotypes.

Asunto(s)

Negro o Afroamericano/genética , Haplotipos/genética , Interleucina-1/genética , Interleucina-6/genética , Linfotoxina-alfa/genética , Población Blanca/genética , Marcadores Genéticos , Genotipo , Humanos , Inflamación , Estados Unidos

RESUMEN

Oxidative stress has been implicated in the pathogenesis of stroke, traumatic brain injuries, and neurodegenerative diseases affecting both neuronal and glial cells in the CNS. In this study we have demonstrated that reactive oxygen species (ROS) dramatically induce the expression of two neuropeptide genes, the opioid proenkephalin (pENK) and the opioid-related proorphanin FQ (pOFQ; also known as pronociceptin) in primary astrocytes. Hydrogen peroxide (H2O2) treatment dose-dependently increased pENK and pOFQ mRNA levels with a maximal effect ( approximately 15-fold increase) being detected at 50 microM concentration. Exposing the astrocyte cultures to hypoxia and subsequent re-oxygenation also led to a profound elevation of pOFQ and pENK mRNA levels. Western blot analysis and immunocytochemistry revealed that H2O2 treatment elicited the phosphorylation and nuclear translocation of ERK 1/2 and p38 MAP kinases. Blockade of the p38 or the ERK MAP kinase pathways (by SB202190 and PD98059, respectively) prevented the H2O2-induced increase in pENK and pOFQ mRNA levels indicating a central role for these cascades in the regulation of pOFQ and pENK genes in response to oxidative stress. Regulation of pOFQ and pENK gene expression by ERK and p38 activation may be mediated through the transcription factor cAMP-response element binding protein (CREB). We observed CREB phosphorylation in response to H2O2, which was also prevented by SB202190 and PD98059. The nuclear factor-kappaB (NF-kappaB) pathway appears to be involved exclusively in the induction of pOFQ transcription by H2O2, as NF-kappaB inhibitors antagonized the effect of oxidative stress on pOFQ, but not on pENK expression. The profound induction of these genes by oxidative stress and these other factors may suggest a role for orphanin FQ and enkephalin in injury and stress responses of the CNS and neuropathophysiological conditions involving reactive oxygen species.

Asunto(s)

Astrocitos/metabolismo , Encefalinas/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/fisiología , Estrés Oxidativo , Precursores de Proteínas/genética , Receptores Opioides/genética , Animales , Transporte Biológico , Western Blotting , Núcleo Celular/enzimología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Activación Enzimática , Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Inmunohistoquímica , FN-kappa B/antagonistas & inhibidores , Fosforilación , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

We have identified the gene encoding nociceptin/orphanin FQ (N/OFQ), the novel opioid-like neuropeptide, as responsive to ciliary neurotrophic factor (CNTF). N/OFQ mRNA levels were induced five- and ninefold by CNTF in striatal and cortical neurons. In primary astrocytes CNTF also increased N/OFQ mRNA levels. CNTF is a multifunctional cytokine that mediates the development and differentiation of both neurons and astrocytes and supports the survival of various neurons. CNTF is also an injury-induced factor in the brain playing a crucial role in astrogliosis. The mechanism by which CNTF elicits these effects is not well understood, but it is likely to involve regulation of specific genes. CNTF regulation of N/OFQ expression was sensitive to the kinase inhibitors H-7 and genistein but not to inhibition of protein synthesis. This pharmacological profile is consistent with CNTF activating the Janus protein tyrosine kinase (JAK)/ signal transducers and activators of transcription (STAT) pathway to induce N/OFQ transcription. In nuclear extracts of CNTF-treated striatal neurons DNA binding of STAT proteins was increased. Radioimmunoassays revealed elevated N/OFQ immunoreactivity in striatal neurons after CNTF treatment. Expression of the related proenkephalin gene was not affected by CNTF in either neuronal or glial cultures. Regulation of N/OFQ expression by CNTF might point to a possible function of N/OFQ during development and after neural injury.

Asunto(s)

Astrocitos/fisiología , Citocinas/fisiología , Regulación de la Expresión Génica/fisiología , Factores de Crecimiento Nervioso/fisiología , Neuronas/fisiología , Péptidos Opioides/genética , Animales , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Factor Neurotrófico Ciliar , Cuerpo Estriado/citología , Cuerpo Estriado/metabolismo , Citocinas/farmacología , ADN/metabolismo , Proteínas de Unión al ADN/genética , Encefalinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Mediadores de Inflamación/farmacología , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/farmacología , Péptidos Opioides/metabolismo , Proteínas Quinasas/fisiología , Precursores de Proteínas/genética , ARN Mensajero/metabolismo , Ratas/embriología , Receptores Opioides mu/genética , Factor de Transcripción STAT1 , Transactivadores/genética , Nociceptina

RESUMEN

There is convincing evidence that genetic factors contribute to the predisposition to alcoholism. In this respect, alcohol-preferring (like C57BL/6 mice) and alcohol-avoiding lines (like DBA/2 mice) of animals served as models in the search for neurobiological substrates of excessive ethanol consumption. One of the systems that is thought to be associated with the incidence of alcoholism is the endogenous opioid system. In the first experiment, basal mRNA levels of mu- and delta-opioid receptors, and of opioid-degrading enzymes enkephalinase (neutral endopeptidase 24.11; NEP) and angiotensin-converting enzyme (ACE) in the brain regions of C57BL/6 and DBA/2 mice did not reveal genetically determined differences in these parameters between the two strains. Furthermore, in the brain regions studied, the corresponding enzyme activities of NEP and ACE did not differ significantly between the lines of mice, except for a higher NEP activity in the striatum and olfactory bulb of DBA/2 mice (p < 0.01). In the second experiment, C57BL/6 and DBA/2 mice were offered a free choice between water and 10% ethanol solution for 4 weeks and were killed thereafter; from another group, ethanol was removed for 3 days and from a third group ethanol was removed for 3 weeks before killing. In the striatum, a highly significant increase in the ACE mRNA amount was detected after 3 weeks of removal of ethanol in C57BL/6 mice, whereas in DBA/2 mice the delta-opioid receptor mRNA level was increased at this time when compared with the corresponding ethanol treatment group. The most striking changes were seen in the hypothalamus, where mu-opioid receptor, ACE, and NEP mRNA amounts markedly decreased after ethanol treatment in both strains. Thus, chronic ethanol intake caused significant changes in the gene expression of distinct components of the endogenous opioid system. These findings further underline an involvement of the opioid system in the effects of ethanol.

Asunto(s)

Consumo de Bebidas Alcohólicas/genética , Alcoholismo/genética , Genotipo , Neprilisina/genética , Peptidil-Dipeptidasa A/genética , Receptores Opioides/genética , Consumo de Bebidas Alcohólicas/efectos adversos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Neprilisina/efectos de los fármacos , Peptidil-Dipeptidasa A/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Receptores Opioides/efectos de los fármacos

RESUMEN

The regulation of nociceptin/orphanin FQ (N/OFQ) gene expression by neuronal activity and by activation of the cyclic AMP signaling pathway in primary neuronal and astroglial cultures is described. Neuronal activity mimicked by veratridine-mediated depolarization profoundly increased N/OFQ gene expression in primary striatal neurons. Calcium entry through L-type, but not N-type, voltage-sensitive calcium channels activated by depolarization appears to be involved, because nitrendipine and nifedipine, but not omega-conotoxin, reduced the induction of N/OFQ expression by veratridine. A selective inhibitor of calcium/calmodulin kinases (KN-62) also antagonized the depolarization-induced increase in N/OFQ mRNA levels, suggesting a role for these enzymes in the activity-dependent induction of N/OFQ gene expression. Constitutively expressed transcription factors may mediate N/OFQ gene expression levels, because veratridine induction of N/OFQ transcription was insensitive to the protein synthesis inhibitor cycloheximide. Regulation of N/OFQ gene expression by depolarization and cyclic AMP is not restricted to striatal neurons, because similar regulation was also observed in neuronal cultures derived from the cerebral cortex. Veratridine did not increase N/OFQ mRNA levels in primary astrocyte cultures; however, elevated intracellular cyclic AMP levels lead to a dramatic, 30-fold induction of N/OFQ mRNA levels in these cells.

Asunto(s)

Astrocitos/enzimología , AMP Cíclico/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas Quinasas Activadas por Mitógenos , Neuronas/enzimología , Péptidos Opioides/genética , Animales , Bucladesina/farmacología , Calcio/metabolismo , Calcio/farmacología , Canales de Calcio/fisiología , Canales de Calcio Tipo L , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Cuerpo Estriado/citología , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas del Tejido Nervioso/fisiología , ARN Mensajero/análisis , Ratas , Receptores Opioides mu/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Nociceptina

RESUMEN

Regulation of gene expression is one of the mechanisms by which neuronal activity elicits long-term changes in neuronal phenotype and function. Although activity-dependent induction of immediate-early genes has been extensively studied, much less is known about the late-response genes. We have investigated the activity-dependent regulation of delta-opioid receptor (DOR) mRNA levels in NG108-15 cells. Transsynaptic activation was mimicked by depolarization with 55 mM KCl or veratridine. Both treatments lead to a time-dependent increase of DOR mRNA levels. Ca2+ entry through L-type voltage-dependent Ca2+ channels activated by depolarization appears to be involved, because L-type channel blockers reduced the induction of DOR expression. Ca2+ binding to calmodulin is the next step in the signal transduction pathway, because a calmodulin antagonist, W7, reduced the effect of veratridine. A selective inhibitor of calmodulin kinases (KN-62) and cyclosporin, an inhibitor of calcineurin, also antagonized the depolarization-induced increase in DOR mRNA levels, which indicates that both calcium/calmodulin-dependent enzymes are involved in the activity-dependent induction of DOR gene expression. Induction of DOR gene expression by an activity-dependent increase in intracellular Ca2+ concentration may serve as a feedback regulatory mechanism because activation of DOR leads to hyperpolarization and lower excitability of neurons.

Asunto(s)

Calcio/fisiología , Calmodulina/fisiología , Regulación de la Expresión Génica/fisiología , Receptores Opioides delta/genética , Transcripción Genética/fisiología , Sitios de Unión/fisiología , Calcineurina/fisiología , Canales de Calcio/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Electrofisiología , Activación Enzimática/fisiología , Membranas/fisiología , ARN Mensajero/metabolismo , Factores de Tiempo , Células Tumorales Cultivadas , Regulación hacia Arriba/fisiología , Veratridina/farmacología

RESUMEN

The effects of activation of the adenylyl cyclase-protein kinase A pathway on the expression of delta-opioid receptor mRNA in the NG108-15 neuroblastoma x glioma cell line has been investigated. Activation of prostaglandin E1 (PGE1) receptors, which are positively coupled to adenylyl cyclase, resulted in a reduction in delta-receptor messenger RNA levels. Direct stimulation of adenylyl cyclase by forskolin or treatment of cells with the cyclic AMP analogue dibutyryl cyclic AMP (db-cAMP) mimicked the effect of PGE1. Down-regulation in receptor protein levels, as measured by loss of radioligand binding sites, was also observed and its extent correlated well with the decrease in the amount of delta-opioid receptor transcripts. D-Ser2-Leu-enkephalin-Thr6 (DSLET) inhibition of adenylyl cyclase activity was also diminished after db-cAMP treatment. Inhibitors of protein kinase A (PKA) partially reversed the PGE1- and db-cAMP-mediated repression of the delta-opioid receptor mRNA levels. The rate of degradation of delta-opioid receptor mRNA in the presence of actinomycin D was not altered in response to db-cAMP, suggesting that mRNA stability is not reduced by PKA action. The regulation of delta-opioid receptor mRNA levels by db-cAMP was not sensitive to the protein synthesis inhibitor cycloheximide, suggesting that de novo protein synthesis is not required in this process.

Asunto(s)

Adenilil Ciclasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Receptores Opioides delta/genética , Inhibidores de Adenilato Ciclasa , Analgésicos/farmacología , Animales , Sitios de Unión/fisiología , Bucladesina/farmacología , Canales de Calcio/fisiología , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Diprenorfina/metabolismo , Diprenorfina/farmacología , Encefalina Leucina/análogos & derivados , Encefalina Leucina/farmacología , Regulación Enzimológica de la Expresión Génica/fisiología , Ratones , Neuroblastoma , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Tritio , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/enzimología

RESUMEN

Competitive polymerase chain reaction assays following reverse transcription have been developed for quantitative analysis of delta and mu opioid receptor gene expression. The assay was used to obtain quantitative measurements of mu and delta opioid receptor expression levels in different brain regions and sensory and sympathetic ganglia in the rat. The assays provide accurate estimates of the relative levels of receptor messenger RNAs by the inclusion in the assays of known amounts of internal standards with the same sequence, except for a small deletion, as the target complementary DNA. The amplification products of target and competitor can be distinguished by size, and their amounts measured by densitometry. Expression of mu and delta opioid receptor messenger RNAs in different regions of the rat brain, somatic and visceral sensory and sympathetic ganglia was investigated using this method. In the brain the highest density of delta receptor messenger RNA was detected in the olfactory bulb, followed by the striatum. The mu receptor was expressed at highest levels in the midbrain-hypothalamic region. All the sensory ganglia studied expressed both mu and delta opioid receptor messenger RNAs. In the nodose ganglion we observed the highest level of mu receptor messenger RNA of any structure studied; in the trigeminal ganglion the level was about 10 times lower than that in the nodose ganglion. Among the dorsal root ganglia, mu receptor messenger RNA density was highest in the lumbar region, followed by the thoracic and cervical regions. The sympathetic superior cervical ganglion expressed a very low level of mu message. Delta receptor messenger RNA was detected only in the sensory ganglia, at levels that were considerably lower than in the striatum. The reverse transcription polymerase chain reaction assay is quantitatively reliable for comparison of messenger RNA levels between different RNA extracts, and sensitive enough to permit the detection and assay of mu and delta opioid receptor gene expression in a single pair of sensory or autonomic ganglia from the rat.

Asunto(s)

Química Encefálica/fisiología , Ganglios/metabolismo , Expresión Génica/fisiología , Receptores Opioides delta/biosíntesis , Receptores Opioides delta/genética , Receptores Opioides mu/biosíntesis , Receptores Opioides mu/genética , Animales , ADN/biosíntesis , Ganglios Espinales/metabolismo , Masculino , Ganglio Nudoso/metabolismo , Reacción en Cadena de la Polimerasa , ARN/biosíntesis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Ganglio Cervical Superior/metabolismo , Ganglio del Trigémino/metabolismo

RESUMEN

Levels of mRNA coding for mu and delta opioid receptors were evaluated by competitive PCR in regions of rat brain where adaptive changes in opioid regulation of adenylyl cyclase occur following chronic morphine treatment. Correlation between receptor protein and mRNA levels after exposure to full and partial agonists were also investigated in NG108-15 cells. Different basal gene expression levels of mu and delta opioid receptors were found in the caudate-putamen, nucleus accumbens, thalamus and periaquaductal gray. After continuous morphine treatment for 7 days both mu and delta opioid receptor mRNA levels were unchanged in all four regions compared with saline-treated controls. In NG108-15 cells marked down-regulation of delta opioid receptors as measured by radioligand binding was observed after 5 or 24 h full agonist (D-Ser3-Leuenkephalin; DSLET) treatment. Morphine, a partial agonist at delta receptors, did not reduce receptor number at either time point. The reduction in delta receptor binding after DSLET treatment was not accompanied by significantly diminished levels of delta receptor mRNA. Unaltered mu and delta receptor mRNA levels in brain after chronic morphine exposure make it unlikely that adaptive changes in the transcription of these genes play a role in the observed tolerance in opioid regulation of adenylyl cyclase.

Asunto(s)

Encéfalo/efectos de los fármacos , Encefalina Leucina/análogos & derivados , Regulación de la Expresión Génica/efectos de los fármacos , ARN Mensajero/biosíntesis , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Animales , Encéfalo/metabolismo , Línea Celular , Encefalina Leucina/farmacología , Código Genético , Masculino , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Receptores Opioides delta/genética , Receptores Opioides mu/genética

RESUMEN

The regulation of mu- (MOR) and delta-opioid receptor (DOR) after chronic cocaine administration has been studied. Male Sprague-Dawley rats were treated for 3 days with saline and cocaine (50 mg/kg/day) delivered by osmotic minipump. Expression of MOR and DOR mRNA in olfactory bulb, nucleus accumbens, and caudate-putamen (caudal and rostral parts) was estimated using quantitative competitive PCR assays after reverse transcription. No changes in the levels of mRNA for DOR were detected after exposure to cocaine in the brain regions examined. A significant increase in the level of MOR mRNA was detected in nucleus accumbens after 3 days of cocaine treatment. In caudate-putamen and olfactory bulb, no change in MOR mRNA was observed after cocaine administration. Both SCH 23390 and eticlopride, selective antagonists of D1- and D2-dopamine receptors, respectively, blocked this cocaine-induced up-regulation of MOR mRNA in nucleus accumbens. We suggest that endogenous opioid systems in nucleus accumbens, the brain region specifically associated with the reinforcing properties of addictive drugs, are regulated by dopaminergic mechanisms and influenced by cocaine treatment.

Asunto(s)

Encéfalo/metabolismo , Cocaína/farmacología , Dopamina/metabolismo , ARN Mensajero/metabolismo , Receptores Opioides delta/genética , Receptores Opioides mu/genética , Animales , Secuencia de Bases , Benzazepinas/farmacología , Antagonistas de Dopamina/farmacología , Masculino , Sondas Moleculares/genética , Datos de Secuencia Molecular , Neostriado/metabolismo , Núcleo Accumbens/metabolismo , Bulbo Olfatorio/metabolismo , Ratas , Ratas Sprague-Dawley , Salicilamidas/farmacología , Factores de Tiempo , Distribución Tisular

RESUMEN

Opioid agonists selective for mu- or delta opioid receptors inhibit adenylyl cyclase in membranes from rat caudate-putamen and nucleus accumbens. The presence of subtypes of delta opioid receptors has been suggested. In both brain regions we have found that the inhibition of adenylyl cyclase by DPDPE was more readily antagonized by 7-benzylidenenaltrexone (BNTX), than by naltriben. In contrast, the inhibitory effects of deltorphin-II and DSLET were more readily antagonized by naltriben, than by BNTX. Neither naltriben nor BNTX significantly antagonized the effect of a mu selective agonist. These results suggest that inhibition of adenylyl cyclase in caudate-putamen and nucleus accumbens is regulated by two forms of delta-opioid receptor with ligand selectivities similar to those two forms proposed to mediate analgesic effect.

Asunto(s)

Adenilil Ciclasas/fisiología , Núcleo Caudado/metabolismo , Núcleo Accumbens/metabolismo , Receptores Opioides delta/fisiología , Inhibidores de Adenilato Ciclasa , Analgésicos/farmacología , Animales , Compuestos de Bencilideno/farmacología , Núcleo Caudado/efectos de los fármacos , Encefalina D-Penicilamina (2,5) , Encefalina Leucina/análogos & derivados , Encefalina Leucina/farmacología , Encefalinas/farmacología , Masculino , Naltrexona/análogos & derivados , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Núcleo Accumbens/efectos de los fármacos , Oligopéptidos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Opioides delta/clasificación , Receptores Opioides delta/efectos de los fármacos

RESUMEN

The regulation of adenylyl cyclase by opioid receptor types was characterized in the rat nucleus accumbens, a brain region that is involved in the reinforcing effects of drugs of abuse, and in the caudate putamen, a region not implicated in drug reinforcement. Both mu and delta opioid ligands inhibited adenylyl cyclase activity in the nucleus accumbens and in the caudate putamen of rat, whereas the kappa agonist, U69,593 (5 alpha, 7 alpha, 8 alpha)-(+)-N-methyl-N-[7-(pyrrolidinyl)-1-oxaspiro [4,5]dec-8-yl]-benzeneacetamide, was ineffective. The mu agonists, DAMGO and Tyr-D-Arg-Phe-Sar, were more potent inhibitors of the enzyme in caudate putamen than in nucleus accumbens. The delta-selective agonists, DSLET and [D-Ala2]-deltorphin II more potently inhibited adenylyl cyclase in nucleus accumbens than in caudate putamen. Inhibition of the enzyme by DAMGO and Tyr-D-Arg-Phe-Sar was antagonized by the mu-selective competitive antagonist, CTOP D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, and the noncompetitive mu antagonists, beta-funaltrexamine and naloxonazine. Inhibition of adenylyl cyclase activity by the delta-selective ligands, DPDPE, DSLET and [D-Ala2]-deltorphin II was unaffected by these antagonists. Conversely, the delta-selective antagonists, ICI 174,864 N-allyl2-Tyr-(alpha-aminisobutyric acid)2-Phe-Leu-OH and naltrindole, blocked the effects of the delta but not the mu opioid ligands. Adenylyl cyclase activity in nucleus accumbens and in caudate putamen is subject to regulation by both mu and delta opioid receptors.

Asunto(s)

Adenilil Ciclasas/metabolismo , Bencenoacetamidas , Núcleo Caudado/fisiología , Núcleo Accumbens/fisiología , Putamen/fisiología , Receptores Opioides delta/fisiología , Receptores Opioides mu/fisiología , Secuencia de Aminoácidos , Animales , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalina D-Penicilamina (2,5) , Encefalina Leucina/análogos & derivados , Encefalina Leucina/farmacología , Encefalinas/farmacología , Masculino , Datos de Secuencia Molecular , Naloxona/análogos & derivados , Naloxona/farmacología , Naltrexona/análogos & derivados , Naltrexona/farmacología , Oligopéptidos/farmacología , Pirrolidinas/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Somatostatina/análogos & derivados , Somatostatina/farmacología

RESUMEN

The newly synthesized 14-alkoxymetopon derivatives, 14-methoxymetopon, 14-ethoxymetopon, 14-methoxy-5-methyl-morphinone, exhibit high affinity for the naloxone binding sites in rat brain. A substantial decrease in affinity was observed, in the presence of NaCl indicating a high degree of agonist activity. All three 14-alkoxymetopon derivatives displayed high affinity for [3H][D-Ala2,(Me)Phe4,Gly-ol5]enkephalin ([3H]DAMGO) binding sites, much less potency toward delta sites and were the least effective at kappa sites. Isolated tissue studies using the guinea pig ileum preparation confirmed their high agonist potency. Following administration the new compounds produced naloxone reversible antinociceptive effects and were 130-300 times more potent than morphine in the acetic acid induced abdominal constriction model in the mouse, and the hot plate and tail flick tests in the rat. The compounds also produced dose-dependent muscle rigidity, and potentiated barbiturate-induced narcosis. The in vivo apparent pA2 values for naloxone against 14-ethoxymetopon and morphine were similar in analgesia, suggesting an interaction with the same (mu) receptor site. The dependence liability of 14-alkoxymetopon derivatives in the withdrawal jumping test was less pronounced than that of morphine in either rats or mice, similar to tolerance to the their analgesic action. It is concluded that the 14-alkoxymetopon derivatives studied are selective and potent agonists at mu opioid receptors, with reduced dependence liability.

Asunto(s)

Analgésicos/farmacología , Derivados de la Morfina/farmacología , Receptores Opioides/efectos de los fármacos , Analgésicos/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Sitios de Unión , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/metabolismo , Encefalinas/farmacología , Femenino , Cobayas , Masculino , Ratones , Derivados de la Morfina/metabolismo , Naloxona/antagonistas & inhibidores , Oximorfona/análogos & derivados , Ratas , Ratas Sprague-Dawley , Receptores Opioides/metabolismo , Especificidad de la Especie , Trastornos Relacionados con Sustancias

RESUMEN

A radiolabelled form of deltorphin II was synthesized by catalytic tritiation using [p-IPhe3]-deltorphin II as a precursor. The ligand labels rat brain membranes with a Kd value of 1.9 nM, and the Bmax was found to be 92 fmol/mg protein. This new tritiated ligand exhibits high affinity for the delta opioid binding site, whereas its binding to the mu type is weak and extremely low for the kappa type. Mu/delta and kappa/delta selectivity ratios were about 900 and 10,000, respectively. The highly delta selective binding properties of this new radioligand suggest that it could serve as an excellent tool for investigating the delta opioid receptors in various species.

Asunto(s)

Bencenoacetamidas , Encéfalo/metabolismo , Oligopéptidos/metabolismo , Receptores Opioides/metabolismo , Analgésicos/metabolismo , Animales , Membrana Celular/metabolismo , Dihidromorfina/metabolismo , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalina D-Penicilamina (2,5) , Encefalina Leucina/análogos & derivados , Encefalina Leucina/metabolismo , Encefalinas/metabolismo , Indicadores y Reactivos , Marcaje Isotópico/métodos , Cinética , Oligopéptidos/síntesis química , Pirrolidinas/metabolismo , Ratas , Ratas Endogámicas , Receptores Opioides delta , Receptores Opioides kappa , Receptores Opioides mu , Tritio

RESUMEN

Vegetative segregation of a mixed plastid population in protoplast fusion-derived cell lines can be directed by a selection favouring the multiplication of one of the parental plastid types. This report defines some of the critical conditions leading to a homogeneous plastid population in cybrid plants generated by protoplast fusion between Nicotiana plumbaginifolia and an albino and streptomycin-resistant N. tabacum plastid mutant. Light (1,500 lx) conferred a strong selective advantage to chloroplasts versus albino plastids, while the lack of this effect in dim light (300 lx) indicated that a sufficient light intensity is essential to the phenomenon. Selection on streptomycin-containing medium in the dark, however, led to the preferential multiplication of resistant plastids. Streptomycin selection of resistant chloroplasts in the light, consequently, results in a plastid selection of doubled stringency. In another experiment a definite, but leaky, selection for chloroplast recombination (selection for greening on streptomycin-containing medium in dim light) was used to reveal various recombination products. Protoplast fusion in fact resulted in cybrid plants showing only simple chimeric segregation of unchanged parental plastids. These results demonstrate the essential requirement for stringent plastid selection, as defined by cell culture conditions, to precede the formation of shoots expected to possess the desired plastid genetic composition.