RESUMO
The common nonsynonymous variant rs16969968 in the α5 nicotinic receptor subunit gene (CHRNA5) is the strongest genetic risk factor for nicotine dependence in European Americans and contributes to risk in African Americans. To comprehensively examine whether other CHRNA5 coding variation influences nicotine dependence risk, we performed targeted sequencing on 1582 nicotine-dependent cases (Fagerström Test for Nicotine Dependence score⩾4) and 1238 non-dependent controls, with independent replication of common and low frequency variants using 12 studies with exome chip data. Nicotine dependence was examined using logistic regression with individual common variants (minor allele frequency (MAF)⩾0.05), aggregate low frequency variants (0.05>MAF⩾0.005) and aggregate rare variants (MAF<0.005). Meta-analysis of primary results was performed with replication studies containing 12 174 heavy and 11 290 light smokers. Next-generation sequencing with 180 × coverage identified 24 nonsynonymous variants and 2 frameshift deletions in CHRNA5, including 9 novel variants in the 2820 subjects. Meta-analysis confirmed the risk effect of the only common variant (rs16969968, European ancestry: odds ratio (OR)=1.3, P=3.5 × 10(-11); African ancestry: OR=1.3, P=0.01) and demonstrated that three low frequency variants contributed an independent risk (aggregate term, European ancestry: OR=1.3, P=0.005; African ancestry: OR=1.4, P=0.0006). The remaining 22 rare coding variants were associated with increased risk of nicotine dependence in the European American primary sample (OR=12.9, P=0.01) and in the same risk direction in African Americans (OR=1.5, P=0.37). Our results indicate that common, low frequency and rare CHRNA5 coding variants are independently associated with nicotine dependence risk. These newly identified variants likely influence the risk for smoking-related diseases such as lung cancer.
Assuntos
Negro ou Afro-Americano/genética , Predisposição Genética para Doença , Proteínas do Tecido Nervoso/genética , Receptores Nicotínicos/genética , Tabagismo/etnologia , Tabagismo/genética , População Branca/genética , Adulto , Feminino , Variação Genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
A single nucleotide polymorphism (SNP) in CHRNA5 (rs16969968, change from an aspartic acid [D] to asparagine [N] at position 398 of the human α5 nicotinic acetylcholine receptor subunit) has been associated with increased risk for nicotine dependence. Consequently, carriers of the risk variant may be at elevated risk for in utero nicotine exposure. To assess whether this gene-environment interaction might impact nicotine intake in developmental nicotine-exposed offspring, we utilized a mouse expressing this human SNP. D and N dams drank nicotine (100 µg/mL) in 0.2% saccharin water or 0.2% saccharin water alone (vehicle) as their sole source of fluid from 30 days prior to breeding until weaning of offspring. The nicotine (D Nic, N Nic) or vehicle (D Veh, N Veh) exposed offspring underwent a 2-bottle choice test between postnatal ages of 30 to 46 days. N Nic offspring consumed the most nicotine at the highest concentration (400 µg/mL) compared with all other groups. In contrast, D Nic offspring drank the least amount of nicotine at all concentrations tested. Nicotine-stimulated dopamine (DA) release measured from striatal synaptosomes was increased in D Nic offspring, while decreased in N Nic offspring relative to their genotype-matched controls. These data suggest that the α5 variant influences the effect of developmental nicotine exposure on nicotine intake of exposed offspring. This gene-environment interaction on striatal DA release may provide motivation for increased nicotine seeking in N Nic offspring and reduced consumption in D Nic offspring.
Assuntos
Nicotina/administração & dosagem , Efeitos Tardios da Exposição Pré-Natal/genética , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Tabagismo/genética , Animais , Modelos Animais de Doenças , Feminino , Interação Gene-Ambiente , Masculino , Camundongos , Camundongos Transgênicos , Nicotina/toxicidade , Polimorfismo de Nucleotídeo Único/genética , GravidezRESUMO
Several studies have demonstrated that there are genetic influences on free-choice oral nicotine consumption in mice. In order to establish the genetic architecture that underlies individual differences in free-choice nicotine consumption, quantitative trait loci (QTL) mapping was used to identify chromosomal regions that influence free-choice nicotine consumption in male and female F(2) mice derived from a cross between C57BL/6J and C3H/HeJ mice. These two mouse strains were chosen not only because they differ significantly for oral nicotine consumption, but also because they are at or near phenotypic extremes for all measures of nicotine sensitivity that have been reported. A four-bottle choice paradigm was used to assess nicotine consumption over an 8-day period. The four bottles contained water or water supplemented with 25, 50 or 100 microg/ml of nicotine base. Using micrograms of nicotine consumed per milliliter of total fluid consumed per day as the nicotine consumption phenotype, four significant QTL were identified. The QTL with the largest LOD score was located on distal chromosome 1 (peak LOD score = 15.7). Other chromosomes with significant QTL include central chromosome 4 (peak LOD score = 4.1), proximal chromosome 7 (peak LOD score = 6.1) and distal chromosome 15 (peak LOD score = 4.8). These four QTL appear to be responsible for up to 62% of the phenotypic variance in oral nicotine consumption.
Assuntos
Comportamento de Escolha/efeitos dos fármacos , Mapeamento Cromossômico , Ingestão de Líquidos/efeitos dos fármacos , Nicotina/administração & dosagem , Locos de Características Quantitativas/genética , Tabagismo/genética , Administração Oral , Animais , Comportamento de Escolha/fisiologia , Cruzamentos Genéticos , Ingestão de Líquidos/fisiologia , Feminino , Escore Lod , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Agonistas Nicotínicos/administração & dosagem , Autoadministração , Fatores Sexuais , Especificidade da EspécieRESUMO
Nicotinic acetylcholine receptors are ligand-gated ion channels that exogenously bind nicotine. Nicotine produces rewarding effects by interacting with these receptors in the brain's reward system. Unlike other receptors, chronic stimulation by an agonist induces an upregulation of receptor number that is not due to increased gene expression in adults; while upregulation also occurs during development and adolescence there have been some opposing findings regarding a change in corresponding gene expression. These receptors have also been well studied with regard to human genetic associations and, based on evidence suggesting shared genetic liabilities between substance use disorders, numerous studies have pointed to a role for this system in comorbid drug use. This review will focus on upregulation of these receptors in adulthood, adolescence and development, as well as the findings from human genetic association studies which point to different roles for these receptors in risk for initiation and continuation of drug use.
Assuntos
Encéfalo/metabolismo , Neurogênese , Receptores Nicotínicos/genética , Tabagismo/metabolismo , Regulação para Cima , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Humanos , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismoRESUMO
In a recent study, we reported that a restriction fragment length polymorphism associated with the alpha4 nicotinic receptor gene (Chrna4) may play a role in regulating differential sensitivity of LS and SS mouse lines to the seizure-inducing effects of nicotine. Since the alpha4 subunit (CHRNA4) is often found as a heteromer with the beta2 subunit (CHRNB2), alpha4 and beta2 cDNAs from the LS and SS mice were cloned and sequenced. A polymorphism in the coding portion of the alpha4 gene was found (1587A to G) which should result in a threonine/alanine substitution at position 529 (T529A). The LS and SS beta2 nicotinic receptor subunit cDNAs were identical. The potential consequences of the alpha4 polymorphism were evaluated using an ion (86Rb+) flux assay that likely measures the function of alpha4beta2-type receptors. LS-SS differences in maximal nicotine-stimulated ion flux were seen when bovine serum albumin (BSA) was not included but this difference was not seen when BSA was included in the perfusion buffer. Current evidence suggests that BSA may alter the ratio of nicotinic receptors that are in the ground state and desensitized forms. Thus, it may be that the Chrna4 T529A substitution leads to a difference in the ratio of the two receptor forms which then promotes differences in receptor function, as well as differential behavioural sensitivity to nicotine.
Assuntos
Nicotina/farmacologia , Polimorfismo Genético , Receptores Nicotínicos/genética , Rubídio/farmacocinética , Sono/genética , Sono/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico Ativo/efeitos dos fármacos , Bovinos , DNA Complementar/genética , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Nicotina/toxicidade , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Soroalbumina Bovina , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Tálamo/efeitos dos fármacos , Tálamo/metabolismoRESUMO
Nicotinic acetylcholine receptor function and binding was measured in 12 brain regions from mice differing in beta2 subunit expression. Function was measured by on-line detection of (86)Rb(+) efflux stimulated under conditions that measure two pharmacologically distinct nicotinic responses: (1) stimulation with 10 microM nicotine, a response that is relatively sensitive to inhibition by the antagonist, dihydro-beta-erythroidine (DHbetaE); and (2) stimulation with 10 microM epibatidine in the presence of 2 microM DHbetaE, a response that is relatively resistant to inhibition by DHbetaE. Deletion of the beta2 subunit profoundly reduced both DHbetaE-sensitive and -resistant (86)Rb(+) efflux in each brain region and essentially eliminated activity in regions such as cerebral cortex and thalamus. However, residual activity was observed in regions such as olfactory bulbs and inferior colliculus. [(3)H]Epibatidine binding was measured under conditions that allow estimation of both high- and low-affinity sites. High-affinity sites sensitive to inhibition by the nicotinic agonist, cytisine, were virtually eliminated in every region by the beta2 null mutation. In contrast, only a subset of the high-affinity sites insensitive to inhibition by cytisine were eliminated in beta2 null mutants, suggesting receptor heterogeniety. Similarly, low affinity [(3)H]epibatidine binding was heterogeneous in that a fraction of the sites required the beta2 subunit. Many remaining sites were sensitive to inhibition by alpha-bungarotoxin indicating that a subset of the low affinity [(3)H]epibatidine binding are of the alpha7* subtype. Distinct regional variation was observed among the 12 brain regions. These studies confirm important roles for beta2-containing receptors in mediating pharmacologically distinct functions and as components of several identifiable binding sites.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Agonistas Nicotínicos/farmacologia , Piridinas/metabolismo , Receptores Nicotínicos/genética , Rubídio/metabolismo , Animais , Bungarotoxinas/farmacologia , Feminino , Genótipo , Cinética , Masculino , Camundongos , Potássio/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Radioisótopos de Rubídio , Estimulação Química , Veratridina/farmacologiaRESUMO
Previous studies have shown that adrenalectomy results in a small increase in hippocampal alpha-bungarotoxin binding, whereas seven days of chronic treatment with high doses of corticosterone results in decreases in alpha-bungarotoxin binding in several brain regions. The studies reported here examined the effects of different doses of corticosterone on brain alpha-bungarotoxin binding. C3H mice were adrenalectomized and treated with corticosterone-containing pellets (0.5-60%) for four days. Alpha-Bungarotoxin binding was measured in eight brain regions. Chronic treatment with corticosterone resulted in plasma corticosterone levels ranging from the low levels observed in an unstressed mouse during the daytime to levels significantly above those observed in mice during the night or as a result of stress. Adrenalectomy resulted in small increases in binding in hippocampus which was reversed by low dose corticosterone treatment. Chronic high-dose corticosterone treatment resulted in significant decreases in binding in four of the eight brain regions examined. Similar, but not identical, results were obtained in two other mouse strains (C57BL and DBA/2). These results argue that corticosterone levels play an important role in modulating the level of the brain nicotinic receptors that bind alpha-bungarotoxin with high affinity.
Assuntos
Encéfalo/efeitos dos fármacos , Bungarotoxinas/farmacologia , Corticosterona/farmacologia , Animais , Ligação Competitiva , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Radioimunoensaio , Fatores de TempoRESUMO
Inbred mouse strains have been shown to differ in their levels of brain alpha-bungarotoxin binding. These differences in alpha-bungarotoxin receptors have been shown to correlate with an animal's sensitivity to nicotine-induced seizures. Recent studies have shown that the alpha 7 nicotinic acetylcholine receptor subunit is the major alpha-bungarotoxin binding site in rodent brain. In this report, we examined whether mouse strains that differ in levels of alpha-bungarotoxin binding and sensitivity to nicotine-induced convulsions also differ for the alpha 7 subunit. A full-length murine alpha 7 cDNA was cloned and sequenced and found to be identical to that of a mouse alpha 7 cDNA recently reported. Subsequently, a comparison of alpha 7 cDNA sequences and RNA species was performed between two strains (C3H/2 and DBA/2) that differ in levels of brain alpha-bungarotoxin binding and sensitivity to nicotine-induced seizures. The only difference observed was a single nucleotide difference in the open reading frame of alpha 7 that does not affect the primary amino acid sequence. Inbred strains were also surveyed for restriction fragment length polymorphisms at the alpha 7 locus. Strain-specific polymorphisms were identified, and F2 and backcross animals from a classic genetic cross between C3H/2 and DBA/2 mice were compared for the inheritance of alpha 7 genotype and alpha-bungarotoxin receptor levels. A significant association between genotype and receptor levels was observed in both, the F2 and backcross generations. These results indicate that alpha 7 genotype is an important determinant of alpha-bungarotoxin receptor levels.
Assuntos
Encéfalo/metabolismo , Bungarotoxinas/metabolismo , Ligação Genética , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Polimorfismo de Fragmento de RestriçãoRESUMO
The studies outlined here used pharmacological and genetic approaches to attempt to identify the nicotinic receptors that modulate nicotine-induced seizures. Full-blown clonic-tonic seizures were induced by intracerebroventricular (i.c.v.) injection of nicotine, the alpha4beta2 selective agonist ABT-418 and the alpha7-selective GTS-21. Cytisine, which is a partial agonist at alpha4beta2-type receptors, produced partial seizures. DHbetaE and MLA did not block nicotine-induced seizures. Instead, both antagonists caused seizures. Restriction fragment length polymorphisms (RFLPs) for the alpha7 receptor were identified in two inbred strains (C3H and DBA) that differ in sensitivity to nicotine-induced seizures. F2 mice derived from a C3H x DBA cross that were homozygous for the C3H variant of the alpha7 RFLP were more sensitive to nicotine-induced seizures than were F2 mice that were homozygous for the DBA RFLP. In a study that used RI strains derived from two selectively bred mouse lines (LS and SS), an association between sensitivity to nicotine-induced seizures and an RFLP associated with the alpha4 gene was found. These data support the assertion that both alpha4 and alpha7 receptor types are involved in modulating convulsions produced by nicotine.
Assuntos
Comportamento Animal/fisiologia , Receptores Nicotínicos/genética , Alcaloides/farmacologia , Animais , Azocinas , Comportamento Animal/efeitos dos fármacos , Cruzamentos Genéticos , Relação Dose-Resposta a Droga , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Humanos , Hibridização Genética , Injeções Intraperitoneais , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Polimorfismo de Fragmento de Restrição , Quinolizinas , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/fisiologia , Convulsões/induzido quimicamente , Convulsões/genética , Receptor Nicotínico de Acetilcolina alfa7RESUMO
C3H and DBA/2 mice differ in their hippocampal inhibitory function, as measured by the inhibitory gating of pyramidal neuron response to repeated auditory stimulation. This functional difference appears to be related to differences in expression of the alpha7 nicotinic cholinergic receptor, which may be generally expressed by interneurons. This study examines the relationship between genetic variation in alpha7 receptor subunit expression and GABAergic interneuron distribution in various regions and layers of the hippocampus in the two mouse strains. Subpopulations of hippocampal interneurons in both mouse strains were found to bind [(125)I]alpha-bungarotoxin. However, the distribution of the [(125)I]alpha-bungarotoxin-positive hippocampal interneurons was significantly different between C3H and DBA/2 mice. In region CA1, and to a lesser extent in region CA3, DBA/2 mice had increased numbers of [(125)I]alpha-bungarotoxin-positive neurons in stratum lacunosum-moleculare and decreased numbers in stratum oriens. Similar differences in GABAergic neuron distribution were observed in region CA1 in the two strains. C3H/DBA/2 F1 animals were backcrossed to the C3H parental strain for six generations, with selection for either the DBA/2 or C3H allelic variant of the alpha7 receptor gene. The distribution of [(125)I]alpha-bungarotoxin labeling closely resembled the DBA/2 parental phenotype in animals retaining the DBA/2 allele of the alpha7 gene. These data suggest that the alpha7 receptor gene locus may influence the anatomical organization of at least a subset of hippocampal interneurons by an as yet unidentified mechanism. This difference in interneuron anatomy may also contribute to functional differences in inhibitory sensory gating between the two strains.
Assuntos
Regulação da Expressão Gênica/genética , Hipocampo/crescimento & desenvolvimento , Interneurônios/citologia , Camundongos Endogâmicos C3H/crescimento & desenvolvimento , Camundongos Endogâmicos DBA/crescimento & desenvolvimento , Inibição Neural/genética , Receptores Nicotínicos/genética , Alelos , Animais , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Bungarotoxinas/farmacocinética , Contagem de Células , Feminino , Testes Genéticos , Genótipo , Hipocampo/citologia , Hipocampo/metabolismo , Imuno-Histoquímica , Interneurônios/metabolismo , Radioisótopos do Iodo , Masculino , Camundongos , Camundongos Endogâmicos C3H/anatomia & histologia , Camundongos Endogâmicos C3H/metabolismo , Camundongos Endogâmicos DBA/anatomia & histologia , Camundongos Endogâmicos DBA/metabolismo , Fenótipo , Polimorfismo de Fragmento de Restrição , Ensaio Radioligante , Receptores Nicotínicos/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7 , Ácido gama-Aminobutírico/metabolismoRESUMO
Autoradiographic techniques were used to localize cholinergic receptors in the central nervous system of female DBA mice. Nicotinic receptors were identified using [3H]-L-nicotine and alpha-[125I]-bungarotoxin (BTX); [3H]-quinuclidinyl benzilate (QNB) was used to examine muscarinic receptor binding. There was little overlap between the regional distribution of binding sites for these ligands. Nicotine binding was highest in thalamic nuclei, the superior colliculus and the interpeduncular nucleus. For BTX binding, high density receptor populations were identified in the hippocampus, caudate putamen, colliculi (superior and inferior) and various nuclei in the hypothalamus and hindbrain. Muscarinic receptors were distributed more uniformly than nicotinic receptors; the colliculi, hippocampus and cerebral cortex had the highest level of QNB binding. Species differences between rats and mice in terms of cholinergic receptor binding are discussed.
Assuntos
Encéfalo/metabolismo , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Autorradiografia , Bungarotoxinas/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos DBA , Quinuclidinil Benzilato/metabolismoRESUMO
Nicotine was administered intravenously to DBA mice through cannulae implanted in the jugular veins. Five groups of animals were treated: a control group which received saline and four nicotine treatment groups. All of the nicotine treatment groups received a dose of 4.0 mg/kg/hr. The first group received continuous infusion, the second group received 1 mg/kg pulses four times an hour, the third group received 2 mg/kg pulses twice an hour, and the fourth group received 4 mg/kg pulses once an hour. After a 10-day treatment period, the animals were tested for tolerance to an acute intraperitoneal administration of nicotine. Tolerance was measured using a test battery composed of the following tests: respiratory rate, acoustic startle response, Y-maze crosses and rears, heart rate, and body temperature. Mice from each of the four nicotine treatment groups were tolerant to the acute effect of nicotine, but the extent of tolerance varied among the groups as follows: continuous infusion less than 1 mg/kg pulses four times/hr less than 2 mg/kg pulses twice/hr less than 4 mg/kg pulse once/hr. Chronic nicotine infusion resulted in significant increases in the binding of L-[3H]nicotine in all six brain regions assayed and in significant increases in the binding of alpha-[125I]bungarotoxin binding in cerebral cortex and hippocampus. All increases in binding resulted from increases in Bmax for these ligands. In contrast to the effects observed for tolerance development, the increases in [3H]nicotine binding were not significantly affected by the kinetics of nicotine infusion.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Encéfalo/efeitos dos fármacos , Nicotina/administração & dosagem , Receptores Opioides/metabolismo , Animais , Temperatura Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/fisiologia , Bungarotoxinas/metabolismo , Relação Dose-Resposta a Droga , Tolerância a Medicamentos , Feminino , Frequência Cardíaca/efeitos dos fármacos , Infusões Intravenosas , Cinética , Camundongos , Camundongos Endogâmicos DBA , Nicotina/metabolismo , Reflexo de Sobressalto/efeitos dos fármacos , Respiração/efeitos dos fármacosRESUMO
Male mice from 19 inbred strains were tested for the effects of nicotine on six responses: respiratory rate, acoustic startle response, Y-maze crosses, Y-maze rears, heart rate and body temperature. Dose-response curves were constructed for each strain on each test in a multitest battery. Results indicated that the responses were strongly influenced by the genotype of the animal. Comparison of the results from the six tests measured in this study and the results previously reported for nicotine-induced seizures in these same strains indicated that the responses could be grouped into two major classes: a set characterized by Y-maze crosses, Y-maze rears and body temperature and a set characterized by seizure sensitivity and seizure latency. Responses observed for respiratory rate and startle response shared characteristics with both of these sets, while nicotine effect on heart rate was fairly unique. The results have identified strains of mice which are differentially sensitive to the effects of nicotine.
Assuntos
Camundongos Endogâmicos/genética , Nicotina/farmacologia , Farmacogenética , Estimulação Acústica , Animais , Temperatura Corporal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Frequência Cardíaca/efeitos dos fármacos , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Reflexo de Sobressalto/efeitos dos fármacos , Respiração/efeitos dos fármacos , Especificidade da EspécieRESUMO
A restriction fragment length polymorphism (RFLP) associated with a major nicotinic receptor subunit (i.e., alpha4) has been identified in two mouse lines that were selectively bred for differences in sensitivity to ethanol. These mice, referred to as Long-Sleep (LS) and Short-Sleep (SS) mice, also differ in sensitivity to several effects of nicotine. The potential role of the alpha4 RFLP in regulating several responses to nicotine and ethanol was evaluated by using the LSxSS-derived recombinant inbred (RI) strains. Those RI strains that carried the LS-like alpha4 RFLP were more sensitive to the depressant effects of nicotine on Y-maze crossing and rearing activities and ethanol-induced increases in Y-maze crossing activity than were those RI strains that carry the SS-like alpha4 RFLP. The LS-like RI strains were also more sensitive to nicotine-induced hypothermia. The RFLP was not associated with strain differences in ethanol-induced body temperature or sleep time. The potential role of the RFLP in regulating ethanol and nicotine consumption was evaluated in heterogeneous stock (HS) mice. An association was found between the alpha4 RFLP and variation in ethanol consumption, but not in nicotine consumption, as measured in a four-bottle choice test. Recent studies of ethanol and tobacco abuse by human beings suggest that common genes may influence these two forms of substance abuse. The results of the studies reported here suggest that the alpha4 nicotinic receptor gene should be evaluated for its potential role in regulating ethanol and tobacco abuse in human beings.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/fisiologia , Consumo de Bebidas Alcoólicas/genética , Animais , Comportamento Animal/efeitos dos fármacos , Temperatura Corporal/efeitos dos fármacos , Comportamento de Escolha/efeitos dos fármacos , Suscetibilidade a Doenças , Resistência a Medicamentos , Feminino , Hipotermia/induzido quimicamente , Injeções Intraperitoneais , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos/genética , Camundongos Endogâmicos/fisiologia , Atividade Motora/efeitos dos fármacos , Polimorfismo de Fragmento de Restrição , Receptores Nicotínicos/genética , Recombinação Genética , Sono/efeitos dos fármacos , Sono/fisiologia , Fumar/genética , Fatores de TempoRESUMO
Smoking behavior is a complex, which includes multiple stages in the progression from experimentation to continued use and dependence. The experience of subjective effects, such as dizziness, euphoria, heart pounding, nausea and high, have been associated with varying degrees of persistence and subsequent abuse/dependence of marijuana, cocaine, tobacco and alcohol (Grant et al. 2005, Wagner & Anthony 2002). Previous studies have reported associations between neuronal nicotinic receptor (CHRN) genes and subjective effects to nicotine. We sought to replicate and expand this work by examining eight single nucleotide polymorphisms (SNPs) in a sample of adult smokers (n = 316) who reported subjective effects following cigarette smoking in a controlled laboratory environment. Two SNPs each in the CHRNB2, CHRNB3, CHRNA6 and CHRNA4 genes were examined. A significant association was found between two SNPs and physical effects reported after smoking the first experimental cigarette. SNP rs2072658 is upstream of CHRNB2 (P-value = 0.0046) and rs2229959 is a synonymous change in exon 5 of CHRNA4 (P value = 0.0051). We also examined possible functional relevance of SNP rs2072658 using an in vitro gene expression assay. These studies provided evidence that the minor allele of rs2072658 may lead to decreased gene expression, using two separate cell lines, P19 and SH-SY5Y (18% P < 0.001 and 26% P < 0.001 respectively). The human genetic study and functional assays suggest that variation in the promoter region of CHRNB2 gene may be important in mediating levels of expression of the ß2 nicotinic receptor subunit, which may be associated with variation in subjective response to nicotine.
Assuntos
Expressão Gênica/fisiologia , Nicotina/farmacologia , Regiões Promotoras Genéticas/genética , Receptores Nicotínicos/genética , Fumar/genética , Adolescente , Adulto , Células Cultivadas , DNA/genética , Etnicidade , Feminino , Genótipo , Humanos , Luciferases/genética , Masculino , Pessoa de Meia-Idade , Fenótipo , Plasmídeos/genética , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Caracteres Sexuais , Fumar/epidemiologia , Fumar/psicologia , Fatores Socioeconômicos , Transfecção , Adulto JovemRESUMO
Mice of two inbred strains, DBA and C3H, were infused i.v. with saline, or 2, 4 or 6 mg/kg/hr of nicotine for 10 days. Two hours after termination of infusion the mice were challenged with one of several nicotine doses and the effects on respiration rate, Y-maze activity and rears, acoustic startle response, heart rate and body temperature were measured. Saline-infused C3H mice were less responsive than were DBA mice for all these tests with the exception of the acoustic startle response test. Nicotine-treated DBA mice developed a dose-related tolerance for most measures, whereas C3H mice did not appear to develop tolerance to any measure until the infusion dose reached 4 mg/kg/hr. The two mouse strains did not differ in the number of [3H] nicotine binding sites in six brain regions, and chronic nicotine treatment elicited similar changes in the binding of this ligand in the two strains. C3H mice had greater concentrations of alpha-[125I] BTX binding in hippocampus, midbrain and hypothalamus than did DBA mice. Chronic nicotine treatment resulted in an identical increase in bungarotoxin binding in the two mouse strains such that the initial strain differences were maintained. These results indicate that a threshold in drug response must be surpassed before a mouse develops tolerance to nicotine. In addition, mechanisms other than differences in receptor numbers must be invoked to explain differences in response to nicotine.
Assuntos
Camundongos Endogâmicos C3H/metabolismo , Camundongos Endogâmicos DBA/metabolismo , Nicotina/farmacologia , Receptores Nicotínicos/metabolismo , Animais , Temperatura Corporal , Encéfalo/efeitos dos fármacos , Bungarotoxinas/metabolismo , Relação Dose-Resposta a Droga , Tolerância a Medicamentos , Feminino , Frequência Cardíaca/efeitos dos fármacos , Camundongos , Consumo de Oxigênio/efeitos dos fármacos , Reflexo de Sobressalto/efeitos dos fármacosRESUMO
Restriction fragment length polymorphisms (rflps) have been identified for the nicotinic ACh receptor subunit genes alpha 5 and alpha 7 between two mouse strains (C3H/2ibg and DBA/2ibg) that differ in sensitivity to the convulsant effects of nicotine. In the study reported here, F2 animals derived from these two parental stains were tested for their sensitivity to the convulsant effects of nicotine as measured by seizure frequency and overall sensitivity score. Subsequently, the animals were genotyped for the alpha 5 and alpha 7 rflps. In addition, levels of alpha-bungarotoxin (alpha-BTX) binding were measured in four brain regions (colliculi, hippocampus, hypothalamus and striatum) to determine whether there is a correlation among alpha-BTX binding levels, sensitivity to nicotine and nicotinic ACh receptor subunit genotype. A significant relationship was observed between alpha 5 and alpha 7 genotype and sensitivity to nicotine. In addition, the alpha 7 rflp significantly correlated with levels of alpha-BTX binding in hippocampus, colliculi and striatum. The alpha 5 rflp did not correlate with alpha-BTX binding levels in any brain region. Levels of alpha-BTX binding did not correlate with nicotine-induced seizure sensitivity or overall nicotine sensitivity score in any of the four brain regions examined.
Assuntos
Nicotina/farmacologia , Receptores Nicotínicos/genética , Convulsões/induzido quimicamente , Animais , Encéfalo/metabolismo , Bungarotoxinas/metabolismo , Genótipo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos DBA , Polimorfismo Genético , Especificidade da EspécieRESUMO
The experiments reported here examined the time course of the development and loss of tolerance to nicotine as well as the time course for the up-regulation and return to basal levels of brain nicotinic receptors. Nicotine was administered by continuous i.v. infusion through cannulae implanted in the right jugular veins of DBA mice. Tolerance to the effects of nicotine on Y-maze activity and rears, body temperature and heart rate was seen accompanying the infusion of 4 mg/kg/hr of nicotine. Maximal tolerance was obtained in 4 days and was paralleled by increases in the number of brain nicotine binding sites. The binding of alpha-bungarotoxin was also increased by chronic nicotine treatment but increases in binding preceded the development of detectable tolerance to the challenge dose of nicotine. Chronic nicotine treatment had no effect on quinuclidinyl benzilate binding. Tolerance to the effects of nicotine was lost at different rates for the various tests in that base-line response was attained for the Y-maze activity and rearings test in 8 days, whereas 12 to 16 days were required to regain control response for the body temperature test. Tolerance for the heart rate test persisted throughout the 20-day withdrawal period. Brain nicotine binding sites had returned to control levels by 8 days after treatment whereas the alpha-bungarotoxin binding sites were at control levels at the earliest time of postinfusion testing (4 days). Brain quinuclidinyl benzilate binding sites were unaffected by nicotine treatment and did not change during the withdrawal period. The changes in nicotine binding in the various brain regions during the onset and offset experiments were virtually identical. These changes in binding correlated highly with the acquisition and loss of tolerance to the effects of nicotine on the Y-maze locomotor activity and body temperature tests. The correlations between changes in nicotine binding and the Y-maze rearings and the heart rate tests were lower but some association is suggested. Although changes in nicotine binding may explain some of the tolerance observed, other explanations for tolerance must be sought as well.
Assuntos
Química Encefálica/efeitos dos fármacos , Nicotina/farmacologia , Receptores Nicotínicos/análise , Animais , Temperatura Corporal/efeitos dos fármacos , Bungarotoxinas/metabolismo , Tolerância a Medicamentos , Feminino , Camundongos , Camundongos Endogâmicos DBA , Atividade Motora/efeitos dos fármacos , Receptores Nicotínicos/efeitos dos fármacos , Síndrome de Abstinência a Substâncias/metabolismo , Fatores de TempoRESUMO
Two distinct binding sites with properties corresponding to those expected for nicotinic cholinergic receptors can be identified in brain by the specific binding of nicotine (or acetylcholine) and alpha-bungarotoxin. The effects of modification of these binding sites by treatment with the disulfide-reducing agent dithiothreitol were examined in tissue prepared from DBA mouse brains. Treatment with dithiothreitol reduced the binding measured with either ligand, and reoxidization of the disulfides fully restored binding. The effects of dithiothreitol treatment appeared to be due to a reduction in the maximal binding of nicotine and to a decrease in the binding affinity for alpha-bungarotoxin. Agonist affinity for the alpha-bungarotoxin binding site was reduced by treatment with low concentrations of dithiothreitol. The nicotine binding sites remaining after disulfide treatment displayed rates of ligand association and dissociation similar to those of unmodified tissue, but treatment of previously unmodified tissue with dithiothreitol accelerated the rate of nicotine dissociation. After reduction, both binding sites could be selectively alkylated with bromoacetylcholine. The results suggest that both putative nicotinic receptors in brain respond similarly to disulfide reduction and that their responses resemble those known for the nicotinic receptor of electric tissue.
Assuntos
Encéfalo/metabolismo , Ditiotreitol/farmacologia , Nicotina/metabolismo , Alquilação , Animais , Sítios de Ligação , Bungarotoxinas/metabolismo , Dissulfetos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos DBA , OxirreduçãoRESUMO
The properties of the binding sites for radiolabeled acetylcholine (measured in the presence of atropine), nicotine, and alpha-bungarotoxin were compared in brain tissue prepared from both rat and mouse. These three binding sites were tested for the following properties: affinity and density of ligand binding, effects of competitive inhibitors, regional distribution, effects of treatment with dithiothreitol and the reversal of these effects by treatment with 5,5'-dithiobis(2-nitrobenzoic acid), thermal lability, effects of protease treatment, and response to chronic administration of nicotine in vivo. The binding sites for acetylcholine and nicotine were affected identically for all measurements, whereas the binding site for alpha-bungarotoxin was affected in a manner different from that for the other two ligands. Although the regional distribution of nicotine and acetylcholine binding differed between rat and mouse brain, other properties of this binding site were very similar between the two species. The results are consistent with the proposal that acetylcholine and nicotine bind to the same sites in both rat and mouse brain, whereas alpha-bungarotoxin binds to different sites.