RESUMO
The clinical overlap between monogenic Familial Hemiplegic Migraine (FHM) and common migraine subtypes, and the fact that all three FHM genes are involved in the transport of ions, suggest that ion transport genes may underlie susceptibility to common forms of migraine. To test this leading hypothesis, we examined common variation in 155 ion transport genes using 5257 single nucleotide polymorphisms (SNPs) in a Finnish sample of 841 unrelated migraine with aura cases and 884 unrelated non-migraine controls. The top signals were then tested for replication in four independent migraine case-control samples from the Netherlands, Germany and Australia, totalling 2835 unrelated migraine cases and 2740 unrelated controls. SNPs within 12 genes (KCNB2, KCNQ3, CLIC5, ATP2C2, CACNA1E, CACNB2, KCNE2, KCNK12, KCNK2, KCNS3, SCN5A and SCN9A) with promising nominal association (0.00041 < P < 0.005) in the Finnish sample were selected for replication. Although no variant remained significant after adjusting for multiple testing nor produced consistent evidence for association across all cohorts, a significant epistatic interaction between KCNB2 SNP rs1431656 (chromosome 8q13.3) and CACNB2 SNP rs7076100 (chromosome 10p12.33) (pointwise P = 0.00002; global P = 0.02) was observed in the Finnish case-control sample. We conclude that common variants of moderate effect size in ion transport genes do not play a major role in susceptibility to common migraine within these European populations, although there is some evidence for epistatic interaction between potassium and calcium channel genes, KCNB2 and CACNB2. Multiple rare variants or trans-regulatory elements of these genes are not ruled out.
Assuntos
Genes/genética , Transporte de Íons/genética , Enxaqueca sem Aura/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Criança , Demografia , Feminino , Finlândia , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , População Branca/genética , Adulto JovemRESUMO
Genetic variation may partially underlie complex personality and physiological traits--such as impulsivity, risk taking and stress responsivity--as well as a substantial proportion of vulnerability to addictive diseases. Furthermore, personality and physiological traits themselves may differentially affect the various stages of addiction, defined chronologically as initiation of drug use, regular drug use, addiction/dependence and potentially relapse. Here we focus on recent approaches to the study of genetic variation in these personality and physiological traits, and their influence on and interaction with addictive diseases.
Assuntos
Comportamento Impulsivo/genética , Assunção de Riscos , Estresse Fisiológico/genética , Transtornos Relacionados ao Uso de Substâncias/genética , Animais , Comorbidade , Variação Genética , Humanos , Modelos Biológicos , Personalidade/genética , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Transtornos Relacionados ao Uso de Substâncias/fisiopatologiaRESUMO
Catechol-O-methyltransferase (COMT) catalyzes the breakdown of catechol neurotransmitters, including dopamine, which plays a prominent role in drug reward. A common single nucleotide polymorphism (SNP), G472A, codes for a Val158Met substitution and results in a fourfold down regulation of enzyme activity. We sequenced exon IV of COMT gene in search for novel polymorphisms and then genotyped four out of five identified by direct sequencing, using TaqMan assay on 266 opioid-dependent and 173 control subjects. Genotype frequencies of the G472A SNP varied significantly (P = 0.029) among the three main ethnic/cultural groups (Caucasians, Hispanics, and African Americans). Using a genotype test, we found a trend to point-wise association (P = 0.053) of the G472A SNP in Hispanic subjects with opiate addiction. Further analysis of G472A genotypes in Hispanic subjects with data stratified by gender identified a point-wise significant (P = 0.049) association of G/A and A/A genotypes with opiate addiction in women, but not men. These point-wise significant results are not significant experiment-wise (at P < 0.05) after correction for multiple testing. No significant association was found with haplotypes of the three most common SNPs. Linkage disequilibrium patterns were similar for the three ethnic/cultural groups.
Assuntos
Catecol O-Metiltransferase/genética , Hispânico ou Latino/genética , Transtornos Relacionados ao Uso de Opioides/etnologia , Transtornos Relacionados ao Uso de Opioides/genética , Polimorfismo de Nucleotídeo Único , Negro ou Afro-Americano/genética , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/fisiologia , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Frequência do Gene , Genótipo , Humanos , Isoenzimas/genética , Desequilíbrio de Ligação , Metionina/genética , Transtornos Relacionados ao Uso de Opioides/enzimologia , Polimorfismo de Nucleotídeo Único/fisiologia , Fatores Sexuais , Valina/genética , População Branca/genéticaRESUMO
Addiction to drugs, such as heroin, cocaine and alcohol, exacts great human and financial costs on society, but the development of pharmacotherapies for addiction has been largely neglected by the pharmaceutical industry. With advances in our understanding of the underlying biology of addictions now opening the door for the development of novel pharmacotherapies, it could be time for a reassessment of involvement in this increasingly important therapeutic area. Here, we summarize the current approved and implemented pharmacotherapeutic approaches to the treatment of addiction, and then highlight the most promising areas for future drug development from the perspective of our laboratory and our National Institutes of Health (NIH) National Institute on Drug Abuse (NIDA) Research Center.
Assuntos
Alcoolismo/tratamento farmacológico , Buprenorfina/uso terapêutico , Metadona/uso terapêutico , Naltrexona/uso terapêutico , Antagonistas de Entorpecentes/uso terapêutico , Receptores Opioides mu/agonistas , Transtornos Relacionados ao Uso de Substâncias/tratamento farmacológico , Humanos , Transtornos Relacionados ao Uso de Substâncias/psicologiaRESUMO
The mu opioid receptor is centrally involved in the development of the addictive diseases. It also modulates the stress responsive hypothalamic-pituitary-adrenal axis. Receptors encoded by the variant 118G polymorphism in exon 1 of the mu opioid receptor gene have a threefold increase in beta-endorphin binding and beta-endorphin is three times more potent in receptor-mediated activation of G protein-coupled inwardly rectifying potassium channels. Humans with this variant have increased stress response following opioid antagonism. Here, we study basal levels of adrenocorticotropic hormone and cortisol in subjects with this variant. In all, 59 healthy adults were genotyped and had morning levels of adrenocorticotropic hormone and cortisol measured following intravenous administration of saline placebo. Subjects with a 118G allele had significantly greater levels of cortisol than subjects with the prototype gene. Groups did not differ in levels of adrenocorticotropic hormone. A planned comparison revealed significantly greater cortisol in females with at least one copy of the 118G allele compared to females with the prototype gene. There was no significant effect of gender alone, nor was there a significant interaction between gender and genotype, on ACTH or cortisol. Subjects with at least one copy of the 118G allele have increased basal levels of cortisol, which may influence the susceptibility to and treatment of the stress responsive dyscrasia.
Assuntos
Éxons , Hidrocortisona/sangue , Polimorfismo de Nucleotídeo Único , Receptores Opioides mu/genética , Estresse Psicológico/sangue , Estresse Psicológico/genética , Hormônio Adrenocorticotrópico/sangue , Adulto , Área Sob a Curva , Distribuição de Qui-Quadrado , Feminino , Genótipo , Humanos , Masculino , Fatores Sexuais , Cloreto de Sódio/efeitos adversos , Estresse Psicológico/etiologiaRESUMO
The mu-opioid receptor (MOR), through its effects on reward and stress-responsivity, modulates alcohol intake in both animal and human laboratory studies. We have previously demonstrated that the frequently occurring A118G single-nucleotide polymorphism (SNP) in exon 1 of the MORgene (OPRM1), which encodes an amino-acid substitution, is functional and receptors encoded by the variant 118G allele bind the endogenous opioid peptide beta-endorphin with three-fold greater affinity than prototype receptors. Other groups subsequently reported that this variant alters stress-responsivity in normal volunteers and also increases the therapeutic response to naltrexone (a mu-preferring opioid antagonist) in the treatment of alcohol dependence. We compared frequencies of genotypes containing an 118G allele in 389 alcohol-dependent individuals and 170 population-based controls without drug or alcohol abuse or dependence. The A118G SNP was present in the Hardy-Weinberg equilibrium with an overall frequency of the 118G allele of 10.9%. There was a significant overall association between genotypes with an 118G allele and alcohol dependence (p=0.0074). The attributable risk for alcohol dependence in subjects with an 118G allele was 11.1%. There was no difference in A118G genotype between type 1 and type 2 alcoholics. In central Sweden, the functional variant 118G allele in exon 1 of OPRM1 is associated with an increased attributable risk for alcohol dependence.
Assuntos
Alcoolismo/genética , Polimorfismo Genético/genética , Receptores Opioides mu/genética , Adulto , Idoso , Alcoolismo/epidemiologia , Alcoolismo/psicologia , Etnicidade , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Escalas de Graduação Psiquiátrica , Medição de Risco , Suécia/epidemiologiaRESUMO
The kappa opioid receptor (KOR) plays a role in stress responsivity, opiate withdrawal and responses to cocaine. KOR activation by its endogenous ligand dynorphin A(1-17) decreases basal and drug-induced striatal levels of dopamine. The complete structure of the human KOR gene (hOPRK1) has not been previously determined. This study: (i) characterized the genomic structure of the hOPRK1 gene; (ii) identified single nucleotide polymorphisms (SNPs) in the hOPRK1 gene; and (iii) investigated possible associations of these variants with vulnerability to develop heroin addiction. Analysis of 5'-RACE cDNA clones revealed the presence of a novel exon 1 ranging in length from 167 to 251 nucleotides in the 5' 5'-untranslated region of the hOPRK1 mRNA. We found that the hOPRK1 gene has four major exons and three introns, similar to rodent OPRK1 genes. Direct sequencing of amplified DNA containing all four exons and intron 1 of the hOPRK1 gene were evaluated for polymorphisms in 291 subjects (145 former heroin addicts and 146 controls). Twelve SNPs were identified, nine novel variants and three previously reported SNPs. Using logistic regression with opioid dependence as the dependent variable, the 36G>T SNP exhibited a point-wise significant association (P = 0.016) with disease status. The number of haplotypes seen in the three ethnic groups were nine, six and five for African-Americans, Caucasians, and Hispanics, respectively, with corresponding significance levels for differences in haplotype frequencies between cases and controls of P = 0.0742, 0.1015 and 0.0041. Combining ethnicities by Fisher's method yields an empirical significance level of P = 0.0020.
Assuntos
Haplótipos , Transtornos Relacionados ao Uso de Opioides/genética , Receptores Opioides kappa/genética , Regiões 5' não Traduzidas , Povo Asiático/genética , Sequência de Bases , População Negra/genética , Éxons , Feminino , Frequência do Gene , Variação Genética , Hispânico ou Latino/genética , Humanos , Íntrons , Desequilíbrio de Ligação , Masculino , Dados de Sequência Molecular , New York , Transtornos Relacionados ao Uso de Opioides/etnologia , Seleção de Pacientes , Polimorfismo de Nucleotídeo Único , Receptores Opioides kappa/química , Homologia de Sequência do Ácido Nucleico , População Branca/genéticaRESUMO
Drug addiction is a complex disorder that has a large spectrum of causes. Vulnerability to addiction has been shown in twin studies to have a robust genetic component. This genetic basis for addiction has general and specific components for each drug abused. Although many genes have been implicated in drug addiction, only a handful have either been replicated to have an association or to have an identified functional mechanism related to specific effects of abused drugs. A few selected genetic variants that currently look promising for the study of alcohol, opiate, and cocaine addiction are discussed in this article.
Assuntos
Química Encefálica/efeitos dos fármacos , Predisposição Genética para Doença/genética , Transtornos Relacionados ao Uso de Substâncias/genética , Alcoolismo/genética , Alcoolismo/metabolismo , Alcoolismo/fisiopatologia , Animais , Química Encefálica/genética , Transtornos Relacionados ao Uso de Cocaína/genética , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Genótipo , Humanos , Transtornos Relacionados ao Uso de Opioides/genética , Transtornos Relacionados ao Uso de Opioides/metabolismo , Transtornos Relacionados ao Uso de Opioides/fisiopatologia , Polimorfismo Genético/genética , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Transtornos Relacionados ao Uso de Substâncias/fisiopatologiaRESUMO
The roots of the Laboratory of the Biology of the Addictive Diseases are in the development of methadone maintenance for the treatment of opiate addiction. Methadone maintenance therapy continues to be one of the major effective forms of addiction pharmacotherapy and underscores the importance of biological factors in the physiology and treatment of the addictive diseases. Recent work in the Laboratory has focused on the neurobiological, neurochemical, neuroendocrine and behavioral aspects of addictive diseases (principally cocaine and the opiate addictions), using an interdisciplinary approach. The models we have focused on range from in vitro molecular biology and neuroscience, to in vivo animal models, to experiments in normal human populations and patients with specific addictive diseases, and most recently to the human molecular genetics of different addictive diseases. Two long-term corollary hypotheses have guided the Laboratory's work: (1) That the endogenous opioid peptide/receptor systems play a central role in the addictive states and therefore in their treatment. (2) That atypical responsivity to stressors (e.g., in the hypothalamic-pituitary-adrenal axis) plays a role in vulnerability and relapse to specific addictive diseases. This atypical responsivity may be drug-induced, environmentally acquired, and/or due to genetic variation.
Assuntos
Neurobiologia/história , Neurobiologia/tendências , Transtornos Relacionados ao Uso de Substâncias/história , Animais , Modelos Animais de Doenças , Endorfinas/fisiologia , História do Século XX , Humanos , Entorpecentes/farmacocinética , National Institutes of Health (U.S.) , Receptores Opioides/efeitos dos fármacos , Receptores Opioides/genética , Receptores Opioides/fisiologia , Transtornos Relacionados ao Uso de Substâncias/genética , Transtornos Relacionados ao Uso de Substâncias/psicologia , Estados UnidosRESUMO
It has been demonstrated that the opioid peptide dynorphin plays a role in modulating responses to several psychoactive substances including cocaine. Our laboratory and others have found that mRNA levels of dynorphin in the caudate and putamen are elevated after acute or chronic cocaine exposure in rats. Recently, a 68-base pair (bp) repeat polymorphism within the core promoter region of the human prodynorphin gene has been reported to occur in alleles containing one, two, three, or four copies. This repeat contains a putative AP-1 transcription factor binding site; reporter gene constructs with three or four, but not one or two, copies of the tandem repeats were shown to be associated with increases in transcriptional activation in in vitro cellular assays. We hypothesize that this polymorphism may be associated with individual differences in vulnerability to cocaine dependence or abuse. From an ongoing study of the genetics of addiction, 174 subjects were studied, including individuals with a primary diagnosis (DSM-IV criteria) of cocaine dependence (N = 61) or abuse (N = 22), and controls with no history of any substance dependence or abuse (N = 91). We designed primers for polymerase chain reaction (PCR) to amplify sequences of the promoter region of the prodynorphin gene containing the repeat element. The association of alleles containing three or four repeats with cocaine dependence/abuse was examined. With data stratified by ethnic group, pooled relative risk (RR) with Mantel-Haenszel Chi square was calculated: RR = 0.59 (95% confidence interval 0.37-0.95), chi2 (1) = 4.14, P = 0.042. Our results suggest that this allelic variation at the promoter region of the prodynorphin gene (alleles with three or four repeats), which may result in enhanced transcription of the gene, may contribute to relative protection and decrease individual vulnerability to develop cocaine dependence or abuse.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/genética , Encefalinas/genética , Polimorfismo Genético , Regiões Promotoras Genéticas , Precursores de Proteínas/genética , Alelos , Sequência de Bases , Transtornos Relacionados ao Uso de Cocaína/etnologia , DNA , Encefalinas/fisiologia , Frequência do Gene , Humanos , Dados de Sequência Molecular , Precursores de Proteínas/fisiologia , Sequências Repetitivas de Ácido NucleicoRESUMO
TaqMan is significantly more sensitive than other methods of mRNA quantification and makes possible the simultaneous analysis of numerous genes in small brain regions. This technique was used to quantify levels of mRNAs of 21 genes in tissue extracts from caudate putamen and nucleus accumbens from individual rats after 1 day 'binge' cocaine or saline administration. Expression of glyceraldehyde-3-phosphate-dehydrogenase, cyclophilin and actin mRNAs as well as 18S ribosomal RNA were evaluated for normalization of levels of gene expression.
Assuntos
Encéfalo/metabolismo , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Cocaína/farmacologia , Corantes Fluorescentes , Regulação da Expressão Gênica/genética , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/análise , Animais , Encéfalo/efeitos dos fármacos , Transtornos Relacionados ao Uso de Cocaína/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
TaqMan, a variation of fluorescent PCR, is a powerful tool for gene expression and polymorphism studies. Here we describe the design and evaluation of 27 new TaqMan primer-probe sets for rat genes that play a key role in neural signaling. These newly designed and synthesized probes were tested and then used for quantification of RNA isolated from rat brain. The usual length of common TaqMan probes is 25 bases or less. In these studies we constructed probes with lengths of 25-39 bases to span exon-exon junctions of nucleic acids to avoid the influence of DNA contamination upon the RNA quantification. The specific sequences at these positions required probes of these lengths to optimize hybridization. We found that the relocation of the quencher from the traditional 3' position to an internal one increases the sensitivity of probe up to 30 fold. Substitution of 6-carboxyfluorescein with Alexa Fluor 488 as fluorophore and TAMRA with non-fluorescent quencher dabcyl was also investigated. We also describe the evaluation of part of a newly designed set of 27 TaqMan primer-probes for the measurement of differences in gene expression levels in samples from the caudate putamen region of rat brain after 'binge' paradigm cocaine administration. Cocaine-induced alterations in expression of c-fos and preprodynorphin mRNAs measured by TaqMan were confirmed by ribonuclease protection assay.
Assuntos
Primers do DNA , Corantes Fluorescentes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Núcleo Caudado/química , Núcleo Caudado/efeitos dos fármacos , Núcleo Caudado/metabolismo , Transtornos Relacionados ao Uso de Cocaína/genética , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Expressão Gênica/efeitos dos fármacos , Genes fos/genética , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/genética , Masculino , Neuropeptídeos/genética , Polimorfismo Genético , Putamen/química , Putamen/efeitos dos fármacos , Putamen/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Ribossômico 18S/análise , RNA Ribossômico 18S/metabolismo , Ratos , Receptores de Dopamina D2/genética , Receptores Opioides/genética , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Preprodynorphin mRNA was measured in the nucleus accumbens (NAc) and caudate putamen (CPu) after 3-day 'binge' pattern cocaine administration in C57BL/6J and 129/J mice, strains which differ in behavior and in dopamine increases in the CPu after 'binge' cocaine. In the CPu, there was increased preprodynorphin mRNA in C57BL/6J (P<0.05), but not in 129/J mice, with no differences in the NAc. Thus, 129/J mice are hyporesponsive to the preprodynorphin activating effects of acute 'binge' cocaine in the CPu.
Assuntos
Núcleo Caudado/metabolismo , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Dinorfinas/biossíntese , Putamen/metabolismo , RNA Mensageiro/biossíntese , Animais , Comportamento Animal/efeitos dos fármacos , Núcleo Caudado/efeitos dos fármacos , Cocaína/administração & dosagem , Cocaína/farmacologia , Relação Dose-Resposta a Droga , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Precursores de Proteínas/biossíntese , Putamen/efeitos dos fármacos , Especificidade da EspécieRESUMO
The new Kreek-McHugh-Schluger-Kellogg scale ('KMSK scale') is designed to quantify self-exposure to opiates, cocaine, alcohol, and/or tobacco. Each section of the KMSK scale assesses the frequency, amount, and duration of use of a particular substance during the individual's period of greatest consumption. The scale also assesses the mode of use, whether the substance use is current or past, and whether each substance is the substance of choice. The administration time is under 5 min. In an initial validation study of this scale, 100 human subjects were administered the KMSK scale concurrently with the Structured Clinical Interview for DSM-IV (SCID-I DSM-IV version). The sensitivity and specificity were very good for opiates, cocaine, and alcohol use. In addition, the correlations between KMSK scores and the number of SCID-I criteria items met were excellent for opiates and cocaine and good for alcohol use. Nicotine dependence was not assessed in this study as there is no SCID-I nicotine criteria. These preliminary results show that the KMSK scale may have both construct validity similar to that of other established self-report measures and the potential to be an effective screening instrument for the assessment of a lifetime diagnosis of alcohol, opiate, or cocaine dependence.
Assuntos
Transtornos Relacionados ao Uso de Substâncias/diagnóstico , Adulto , Feminino , Humanos , Masculino , Psicometria , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Preprodynorphin and preproenkephalin are protein precursors from which are derived two classes of opioid neurotransmitter peptides. Dynorphin A((1-17)) is produced by proteolytic processing of prodynorphin, and processing of proenkephalin yields the enkephalin peptides. We report here on the isolation and sequencing of multiple clones for these two mRNAs from a cDNA library. Two cDNA clones of preprodynorphin contained the full-length sequence (2.35 kb) with the primary structure predicted from the guinea pig gene sequence. In contrast, one clone encoded the full-length sequence but also an additional 192 nt at the 5' end. This sequence has high homology to the 5' flanking region of the human preprodynorphin gene, and RNase protection assays demonstrated that in addition to a primary initiation site, transcription of this mRNA is initiated at several sites 160-190 nt 5' with respect to the primary site. This difference may alter translational efficiency or mRNA stability. The sequence of preproenkephalin cDNA clones confirmed the structure predicted from the gene sequence. One clone, however, contained sequences encoded by exons 2 and 3, and initiated within the first intron (intron A) of the gene. We used RNase protection mapping to assess the abundance in the brain and pituitary of preproenkephalin transcripts that initiate within intron A. These studies confirmed that the primary transcription start site is 28 nucleotides downstream from the TATAA site, and that intron A sequences are not present in significant amounts in these tissues.
Assuntos
Dinorfinas/química , Dinorfinas/genética , Encefalinas/química , Encefalinas/genética , Precursores de Proteínas/química , Precursores de Proteínas/genética , RNA Mensageiro/química , Sítio de Iniciação de Transcrição , Animais , Sequência de Bases , Encéfalo/fisiologia , Dinorfinas/metabolismo , Cobaias , Masculino , Dados de Sequência Molecular , Hipófise/fisiologia , Precursores de Proteínas/metabolismo , Processamento Pós-Transcricional do RNARESUMO
Male Hartley guinea pigs were administered i.p. injections of cocaine or saline for 2 or 7 days in a "binge" paradigm. RNA was isolated from dissected brain regions and levels of preproenkephalin mRNA and total RNA were quantified by RNase protection assays. Following 2 days of "binge" cocaine administration, no significant alterations in preproenkephalin mRNA levels were detected in six brain regions. Following 7 days of cocaine administration, however, lower levels of preproenkephalin mRNA were observed in the nucleus accumbens and hypothalamus of cocaine-treated animals and higher levels in the frontal cortex and amygdala. These findings differed from previous studies in the rat, so an additional experiment was performed with animals treated at the 7 day time point. For increased statistical power, data from the two experiments were combined and examined by two-way ANOVAs; in this combined analysis, increases in preproenkephalin mRNA were observed in frontal cortex, amygdala, and hippocampus, decreases were found in the nucleus accumbens and hypothalamus, with no change in thalamus, caudate putamen, or cerebellum. These observed differences between guinea pigs and rats make this species an interesting model for neurobiological studies of cocaine-induced alterations in neuropeptide gene expression in the mammalian brain.
Assuntos
Encéfalo/efeitos dos fármacos , Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Encefalinas/efeitos dos fármacos , Precursores de Proteínas/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Cocaína/administração & dosagem , Inibidores da Captação de Dopamina/administração & dosagem , Esquema de Medicação , Encefalinas/genética , Cobaias , Masculino , Precursores de Proteínas/genética , RNA Mensageiro/análise , Especificidade da EspécieRESUMO
Discovery and characterization of the functional A118G mu-(mu)-opioid receptor variant led to hypotheses, now in part proven, about its role in alterations of endogenous human physiology and in responses to opioid antagonist administration. Differences in cellular expression levels, ligand binding, and signal transduction for variant receptors have been documented in vitro. Human genetic studies also indicate that individuals carrying one or two copies of the 118G allele may have increased risk for opiate and alcohol addictions and that this polymorphism may also explain some of the variability in success of opioid antagonist treatment for alcoholism. Future research will further define the role of the A118G variant in addictive diseases and their treatment, in pain perception and opioid analgesia, and for a myriad of other responses mediated by the mu-opioid receptor.
Assuntos
Polimorfismo Genético , Receptores Opioides mu/genética , Estresse Fisiológico , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Humanos , Conformação Proteica , Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The most common single nucleotide polymorphism in the coding region of the human mu opioid receptor gene is the A118G variant, an adenine to guanine transition at nucleotide position 118 of the coding sequence of the gene. This polymorphism codes for an asparagine to aspartic acid substitution at amino acid 40 in the amino-terminus, thereby removing a potential extracellular glycosylation site. Using in vitro cellular expression assays, this variant has been reported to change binding of the endogenous agonist beta-endorphin and signaling of the receptor following binding of beta-endorphin. Three clinical studies report that A118G genotype affects opioid antagonist-mediated increases in cortisol levels. These studies demonstrate a functional role of this variant in responses to endogenous and exogenous opioids. To further characterize function, we expressed the prototype and variant receptors in two types of cells (human 293 embryonic kidney cells and Syrian hamster adenovirus-12-induced tumor cells). Stable expression of variant and prototype receptors was characterized by differences in levels of cell surface binding capacity (B(max)), forskolin-induced cAMP accumulation, as well as agonist-induced accumulation of cAMP (EC(50)) for several agonists, but not for beta-endorphin. In contrast, transiently expressed variant receptors showed only a minor difference in cell surface binding capacity compared to the prototype, and no differences in cAMP EC(50) values.
Assuntos
Polimorfismo de Nucleotídeo Único/genética , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Analgésicos Opioides/farmacocinética , Animais , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/genética , Linhagem Celular , Membrana Celular/metabolismo , Colforsina/farmacologia , Cricetinae , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacocinética , Fibroblastos/metabolismo , Técnicas de Transferência de Genes , Humanos , Rim/citologia , Rim/metabolismo , Mesocricetus , Receptores Opioides mu/efeitos dos fármacosRESUMO
There is strong evidence for a genetic contribution to individual differences in vulnerability to drug addictions. Studies have shown that the 68-base pair repeat polymorphism in the promoter region of the human prodynorphin gene contains a putative AP-1 binding site, and that three or four repeat copies result in greater transcriptional activation. Here, we report on a separate cohort of 302 subjects ascertained and characterized extensively by Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition and Addiction Severity Index criteria as: (1) a control group of 127 subjects with no history of alcohol or drug abuse or dependence; (3) a case group of 82 with cocaine dependence only; and (3) a case group of 93 with cocaine and alcohol codependence. The promoter region of the prodynorphin gene containing the repeat was amplified from genomic DNA by polymerase chain reaction and analyzed via gel electrophoresis. Statistical tests were performed with data stratified by the three major ethnic groups studied: African American, Caucasian and Hispanic. For analyses, genotypes were grouped into short (1,1; 1,2; 2,2), short/long (1,3; 2,3; 1,4; 2,4) and long (3,3; 3,4; 4,4) repeats. Deviation from Hardy-Weinberg Equilibrium in the African American control group necessitated testing for association using grouped genotypes rather than grouped alleles. In controls, a significant difference was found in grouped genotype distribution among ethnicities. We found a point-wise, but not experiment-wise across-ethnicities, significant difference in grouped genotype frequency between the cocaine/alcohol-codependent group and the controls in African Americans, with genotypes containing longer alleles found at higher frequency in the codependent group.
Assuntos
Alcoolismo/genética , Pareamento de Bases/genética , População Negra/genética , Transtornos Relacionados ao Uso de Cocaína/genética , DNA/genética , Encefalinas/genética , Hispânico ou Latino/genética , Regiões Promotoras Genéticas/genética , Precursores de Proteínas/genética , Sequências de Repetição em Tandem/genética , População Branca/genética , Alelos , Comorbidade , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVES: 5-Hydroxytryptamine (serotonin)-1B receptors (HTR1B) may play an important role in psychiatric disorders and drug and alcohol dependence. In this study we report on genotype, molecular haplotype and statistically estimated haplotype analyses of previously identified polymorphisms in positions -261T>G, -161A>T, 129C>T, 861G>C and 1180A>G of the HTR1B gene in ethnically diverse populations (African-Americans, Caucasians, Hispanics and Asians) including 235 former heroin addicts and 161 control subjects from New York City. The objectives were to test for an association of molecular and statistically estimated haplotypes and genotypes in HTR1B gene with heroin addiction and to compare results provided by molecular and statistically estimated haplotyping methods. METHODS: Genotype analysis was performed using a standard TaqMan protocol. Molecular haplotype analysis of the subset of polymorphisms consisting of -261T>G, -161A>T and 129C>T was performed using a protocol specially designed by our group, using fluorescent PCR. This is based on use of allele-specific primers complementary to flanking polymorphisms and a fluorescently labeled sequence-specific TaqMan probe set complementary to an internal polymorphism of the haplotype region. Every individual's statistically inferred haplotype pair agreed with the individual's haplotype pair determined by molecular haplotyping. RESULTS AND CONCLUSION: A point-wise significant association of haplotype pairs containing allele G at position 1180 with protective effect from heroin addiction in Caucasians was found. A point-wise nominally significant association of allele 1180G with a protective effect from heroin addiction was found in Caucasians. Statistically significant differences across four ethnic groups in control subjects for allelic frequencies of -261T>G and -161A>T were found.