RESUMO
The aim of this study is to reveal the habits of drug abusers in hair samples from drug rehabilitation units in Hong Kong. With the application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology, a total of 1771 hair samples were analyzed during the period of hair testing service (January 2012 to March 2016) provided to 14 drug rehabilitation units including non-governmental organizations (NGOs), rehabilitation centers, and medical clinics. Hair samples were analyzed for abused drugs and their metabolites simultaneously, including ketamine, norketamine, cocaine, benzoylecgonine, cocaethylene, norcocaine, codeine, MDMA, MDA, MDEA, amphetamine, methamphetamine, morphine, 6-acetylmorphine, phencyclidine, and methadone. The results showed that ketamine (77.2%), cocaine (21.3%), and methamphetamine (16.5%) were the frequently detected drugs among those drug abusers, which is consistent with the reported data. In addition, the usage of multiple drugs was also observed in the hair samples. About 29% of drug-positive samples were detected with multiple drug use. Our studies prove that our locally developed hair drug-testing method and service can be a valid tool to monitor the use of abused drugs, and which could facilitate rehabilitation program management.
Assuntos
Cabelo/química , Drogas Ilícitas/análise , Detecção do Abuso de Substâncias/métodos , Cromatografia Líquida , Hong Kong , Humanos , Espectrometria de Massas em TandemRESUMO
Ketamine is one of the most frequent abused drugs in Hong Kong and South-East Asia, and the cases of ketamine abused have been reported worldwide. Hair has been commonly used as a specimen for the proof of chronic drug abused because of its non-invasiveness and long detection windows. The determinations of ketamine in hair with varieties of state-of-the-art instruments and detection methods have been developed in the past decade; however, the cut-off value for ketamine abuser has not been developed according to the international guidelines. The aim of this study is to propose a cut-off value for ketamine in hair by analyzing ketamine and its metabolite norketamine by LC-MS/MS method in a population of ketamine users in Hong Kong. The limit of detection (LOD) and limit of quantification (LOQ) for ketamine and norketamine were 20pg/mg and 100pg/mg, respectively. From 977 ketamine abusers, the cut-off value for ketamine in hair was proposed to be 400pg/mg of hair. This proposed cut-off value is the concentration of hair ketamine when over 90% of samples are being detected with the presence of norketamine, which is a proof of ketamine abuse. This value could be applied as a screening or occupational cut-off for reference.
Assuntos
Cromatografia Líquida , Drogas Ilícitas/análise , Ketamina/análise , Espectrometria de Massas em Tandem , Adolescente , Adulto , Feminino , Cabelo , Hong Kong , Humanos , Masculino , Reprodutibilidade dos Testes , Adulto JovemRESUMO
A microfluidic chip based nano-HPLC coupled to tandem mass spectrometry (nano-HPLC-Chip-MS/MS) has been developed for simultaneous measurement of abused drugs and metabolites: cocaine, benzoylecgonine, cocaethylene, norcocaine, morphine, codeine, 6-acetylmorphine, phencyclidine, amphetamine, methamphetamine, MDMA, MDA, MDEA, and methadone in the hair of drug abusers. The microfluidic chip was fabricated by laminating polyimide films and it integrated an enrichment column, an analytical column and a nanospray tip. Drugs were extracted from hairs by sonication, and the chromatographic separation was achieved in 15 min. The drug identification and quantification criteria were fulfilled by the triple quardropule tandem mass spectrometry. The linear regression analysis was calibrated by deuterated internal standards with all of the R(2) at least over 0.993. The limit of detection (LOD) and the limit of quantification (LOQ) were from 0.1 to 0.75 and 0.2 to 1.25 pg/mg, respectively. The validation parameters including selectivity, accuracy, precision, stability, and matrix effect were also evaluated here. In conclusion, the developed sample preparation method coupled with the nano-HPLC-Chip-MS/MS method was able to reveal the presence of drugs in hairs from the drug abusers, with the enhanced sensitivity, compared with the conventional HPLC-MS/MS.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cabelo/química , Drogas Ilícitas/análise , Microfluídica/métodos , Nanotecnologia/métodos , Detecção do Abuso de Substâncias/métodos , Espectrometria de Massas em Tandem/métodos , Cabelo/metabolismo , Humanos , Drogas Ilícitas/metabolismo , Limite de DetecçãoRESUMO
Acetylcholinesterase (AChE) is responsible for the hydrolysis of the neurotransmitter, acetylcholine, in the nervous system. The functional localization and oligomerization of AChE T variant are depending primarily on the association of their anchoring partners, either collagen tail (ColQ) or proline-rich membrane anchor (PRiMA). Complexes with ColQ represent the asymmetric forms (A(12)) in muscle, while complexes with PRiMA represent tetrameric globular forms (G(4)) mainly found in brain and muscle. Apart from these traditional molecular forms, a ColQ-linked asymmetric form and a PRiMA-linked globular form of hybrid cholinesterases (ChEs), having both AChE and BChE catalytic subunits, were revealed in chicken brain and muscle. The similarity of various molecular forms of AChE and BChE raises interesting question regarding to their possible relationship in enzyme assembly and localization. The focus of this review is to provide current findings about the biosynthesis of different forms of ChEs together with their anchoring proteins.
RESUMO
Acetylcholinesterase (AChE) anchors onto cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric form in vertebrate brain. The assembly of AChE tetramer with PRiMA requires the C-terminal "t-peptide" in AChE catalytic subunit (AChE(T)). Although mature AChE is well known N-glycosylated, the role of glycosylation in forming the physiologically active PRiMA-linked AChE tetramer has not been studied. Here, several lines of evidence indicate that the N-linked glycosylation of AChE(T) plays a major role for acquisition of AChE full enzymatic activity but does not affect its oligomerization. The expression of the AChE(T) mutant, in which all N-glycosylation sites were deleted, together with PRiMA in HEK293T cells produced a glycan-depleted PRiMA-linked AChE tetramer but with a much higher K(m) value as compared with the wild type. This glycan-depleted enzyme was assembled in endoplasmic reticulum but was not transported to Golgi apparatus or plasma membrane.
Assuntos
Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Biocatálise , Galinhas , Estabilidade Enzimática , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Glicosilação , Células HEK293 , Humanos , Camundongos , Polissacarídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Transporte Proteico , Proteínas Recombinantes/metabolismoRESUMO
An effective way to determine the amount of different neurotransmitters is vital to the study of brain function. Here, a highly sensitive HPLC-MS/MS method was developed to simultaneously measure γ-aminobutyric acid, dopamine, epinephrine, norepinepherine, glutamate and serotonin in one sample. The quantification of the neurotransmitters was achieved by a tandem mass spectrometer using the selected reaction monitoring scan mode. The method validation included selectivity, linearity, accuracy, precision, stability, recovery and matrix effect. For the six neurotransmitters, the linear regression analysis was calibrated by deuterated internal standards with a R(2) of over 0.991, and the limit of detection (LOD) and the limit of quantification (LOQ) were from 2.5 to 500 pg/mg and 7.5 to 1000 pg/mg, respectively. This method was employed here to reveal different types and amounts of neurotransmitters simultaneously in adult and embryonic rat brains. Here, the change of dopamine concentration in embryonic and adult brain was from 0.071 to 0.760 ng/mg of brain tissue, GABA was from 207.643 to 445.148 ng/mg, glutamate was from 679.535 to 1408.920 ng/mg, serotonin was from 0.058 to 0.485 ng/mg and norepinepherine was from 0.054 to 0.290 ng/mg. For epinephrine, it was only detected in embryonic stage but not in adult, with the concentration at 0.241 ng/mg.
Assuntos
Química Encefálica , Cromatografia Líquida/métodos , Neurotransmissores/análise , Espectrometria de Massas em Tandem/métodos , Animais , Dopamina/análise , Dopamina/isolamento & purificação , Estabilidade de Medicamentos , Embrião de Mamíferos , Epinefrina/análise , Epinefrina/isolamento & purificação , Ácido Glutâmico/análise , Ácido Glutâmico/isolamento & purificação , Modelos Lineares , Masculino , Neurotransmissores/isolamento & purificação , Norepinefrina/análise , Norepinefrina/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Serotonina/análise , Serotonina/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Ácido gama-Aminobutírico/análise , Ácido gama-Aminobutírico/isolamento & purificaçãoRESUMO
An effective way to reveal the history of drug abuse is to determine the parental drug and its metabolites in hair. Here, a quantitative HPLC-Chip-MS/MS method was developed for simultaneous measurement of ketamine and its metabolite norketamine in human hair. Ketamine and norketamine were extracted from hair by acid hydrolysis, and then enriched by organic solvent extraction. The chromatographic separation was achieved in 15 min, with the drug identification and quantification by a tandem mass spectrometer. The linear regression analysis was calibrated by deuterated internal standards with a R(2) of over 0.996. The limit of detection (LOD) and the limit of quantification (LOQ) for ketamine and norketamine were 0.5 and 1 pg/mg of hair, respectively. The standard curves were linear from the value of LOQ up to 100 pg/mg of hair. The validation parameters including selectivity, accuracy, precision, stability and matrix effect were also determined. In conclusion, this method was able to reveal the present of ketamine and norketamine with less hair from the drug abusers, and which had the sensitivity of â¼1000-fold higher than the conventional method. In addition, the amount of ketamine and norketamine being detected in different hair segments would be useful in revealing the historical record of ketamine uptake in the drug abusers.
Assuntos
Antagonistas de Aminoácidos Excitatórios/análise , Cabelo/química , Ketamina/análogos & derivados , Ketamina/análise , Detecção do Abuso de Substâncias/métodos , Antagonistas de Aminoácidos Excitatórios/química , Toxicologia Forense , Cromatografia Gasosa-Espectrometria de Massas/instrumentação , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Ketamina/química , Limite de Detecção , Estrutura Molecular , Manejo de EspécimesRESUMO
Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE(T) and BChE(T) with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q(N-GPI)). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q(N-GPI) always consist of AChE(T) and BChE(T) homodimers. The dimer formation of AChE(T) and BChE(T) depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal "t-peptides" in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE(T) or BChE(T) homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.
Assuntos
Acetilcolinesterase/biossíntese , Encéfalo/embriologia , Butirilcolinesterase/biossíntese , Galinhas , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Membrana/biossíntese , Complexos Multienzimáticos/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Multimerização Proteica/fisiologia , Regulação para Cima/fisiologia , Acetilcolinesterase/genética , Animais , Encéfalo/citologia , Encéfalo/enzimologia , Butirilcolinesterase/genética , Células Cultivadas , Embrião de Galinha , Proteínas de Membrana/genética , Complexos Multienzimáticos/genética , Mutação , Proteínas do Tecido Nervoso/genética , Peptídeos/genética , Peptídeos/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transmissão Sináptica/fisiologiaRESUMO
In the mammalian brain, acetylcholinesterase (AChE) is anchored in cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor). We present evidence that at least part of the PRiMA-linked AChE is integrated in membrane microdomains called rafts. A significant proportion of PRiMA-linked AChE tetramers from rat brain was recovered in raft fractions; this proportion was markedly higher at low rather than at high concentrations of cold Triton X-100. The detergent-resistant fraction increased during brain development. In NG108-15 neuroblastoma cells transfected with cDNAs encoding AChE(T) and PRiMA, PRiMA-linked G(4) AChE was found in membrane rafts and showed the same sensitivity to cold Triton X-100 extraction as in the brain. The association of PRiMA-linked AChE with rafts was weaker than that of glycosylphosphatidylinositol-anchored G(2) AChE or G(4) Q(N)-H(C)-linked AChE. It was found to depend on the presence of a cholesterol-binding motif, called CRAC (cholesterol recognition/interaction amino acid consensus), located at the junction of transmembrane and cytoplasmic domains of both PRiMA I and II isoforms. The cytoplasmic domain of PRiMA, which differs between PRiMA I and PRiMA II, appeared to play some role in stabilizing the raft localization of G(4) AChE, because the Triton X-100-resistant fraction was smaller with the shorter PRiMA II isoform than that with the longer PRiMA I isoform.
Assuntos
Acetilcolinesterase/metabolismo , Encéfalo/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Prolina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Humanos , Masculino , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de AminoácidosRESUMO
Heat shock response, an induced transcription of a set of genes in response to high temperature, occurs in all organisms. In neurons, the catalytic subunit of acetylcholinesterase (AChE(T)) interacts with proline-rich membrane anchor (PRiMA) to form a globular tetrameric form (G(4) form). In this study, we examined the effects of heat shock on the transcription and protein assembly of AChE(T) in cultured NG108-15 cells. The transcription of AChE(T) was rapidly induced by heat shock at 40 degrees C, reaching a 15-fold increase in 3h and decreasing thereafter. On the other hand, the level of PRiMA mRNA was not affected after the heat shock. In parallel with AChE(T) mRNA, the enzymatic activity of cellular AChE, in terms of G(1) and G(2) forms, was increased after heat shock; however, the PRiMA-linked G(4) remained unchanged. These results suggest that heat shock can induce the expression level of AChE(T) by the regulation of AChE(T) transcripts in NG108-15 cells.
Assuntos
Acetilcolinesterase/genética , Resposta ao Choque Térmico/genética , Ativação Transcricional , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Animais , Biocatálise , Linhagem Celular Tumoral , Temperatura Alta , Proteínas de Membrana/genética , Camundongos , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
Acetylcholinesterase (AChE), a highly polymorphic enzyme with various splicing variants and molecular isoforms, plays an essential role in the cholinergic neurotransmission by hydrolyzing acetylcholine into choline and acetate. The AChE(T) variant is expressed in the brain and muscle: this subunit forms non-amphiphilic tetramers with a collagen tail (ColQ) as asymmetric AChE (A(12) AChE) in muscle, and amphiphilic tetramers with a proline-rich membrane anchor (PRiMA) as globular AChE (G(4) AChE) in the brain and muscle. During the brain development, the expression of amphiphilic G(4) AChE is up regulated and becomes the predominant form of AChE there. This up-regulation of G(4) AChE can be attributed to the increased expressions of both AChE(T) and PRiMA. A significant portion of this membrane-bound G(4) AChE is localized at the membrane rafts of the cell membranes derived from the brain. This raft association could be directed by PRiMA via its CRAC (cholesterol recognition/interaction amino acid consensus) motif and C-terminus. In cultured cortical neurons and muscles, the PRiMA-linked AChE was clustered and partially co-localized with synaptic proteins. The restricted localizations suggest that the raft association of PRiMA-linked AChE could account for its synaptic localization and function.
Assuntos
Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Multimerização Proteica , Sinapses/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Estrutura Quaternária de Proteína , Transporte Proteico , Sinapses/enzimologiaRESUMO
A Radix Notoginseng flavonol glycoside (RNFG), quercetin 3-O-beta-D-xylopyranosyl-beta-D-galactopyranoside, was isolated from roots of Panax notoginseng. Among different biological properties tested, RNFG possessed a strong activity in preventing amyloid-beta (Abeta)-induced cell death. In an in vitro assay, RNFG inhibited the aggregation of Abeta in a dose-dependent manner. Moreover, application of RNFG in cultured cortical neurons, or PC12 cells, reduced the Abeta-induced cell death in time- and dose-dependent manners, with the suppression of Abeta-induced DNA fragmentation and caspase-3 activation. In cultured neurons, the pre-treatment of RNFG abolished the increase of Ca(2+) mobilization triggered by Abeta. The neuroprotective properties of RNFG required a specific sugar attachment within the main chemical backbone because the flavonol backbone by itself did not show any protective effect. In memory impairment experiments using the passive avoidance task, the administration of RNFG reduced brain damage in scopolamine-treated rats. These results therefore reveal a novel function of Radix Notoginseng and its flavonol glycoside that could be very useful in developing food supplements for the prevention, or potential treatment, of Alzheimer's disease.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Flavonóis/farmacologia , Flavonóis/uso terapêutico , Glicosídeos/farmacologia , Glicosídeos/uso terapêutico , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Panax notoginseng , Fitoterapia/métodos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Células Cultivadas , Feminino , Humanos , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Acetylcholinesterase (AChE) is well known to process different molecular forms via the distinct interacting partners. Proline-rich membrane anchor (PRiMA)-linked tetrameric globular AChE (G4 AChE) is mainly found in the vertebrate brain; however, recent studies from our laboratory have suggested its existence at neuromuscular junctions (nmjs). Both muscle and motor neuron express AChE at the nmjs. In muscle, the expression of PRiMA-linked AChE is down-regulated during myogenic differentiation and by motor neuron innervation. As compared with muscle, spinal cord possessed higher total AChE activity and contained PRiMA-linked AChE forms. The spinal cord expression of this form increased during development. More importantly, PRiMA-linked G4 AChE identified as aggregates localized at nmjs. These findings suggest that the restricted localization of PRiMA-linked G4 AChE at the nmjs could be contributed by the pre-synaptic motor neuron and/or the post-synaptic muscle fiber.
Assuntos
Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Junção Neuromuscular/enzimologia , Medula Espinal/enzimologia , Animais , Diferenciação Celular/genética , Neurônios Motores/enzimologia , Neurônios Motores/ultraestrutura , Músculo Esquelético/enzimologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/inervação , Junção Neuromuscular/crescimento & desenvolvimento , Junção Neuromuscular/ultraestrutura , Terminações Pré-Sinápticas/enzimologia , Terminações Pré-Sinápticas/ultraestrutura , Conformação Proteica , RNA Mensageiro/metabolismo , Ratos , Agregação de Receptores/fisiologia , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/ultraestrutura , Membranas Sinápticas/enzimologia , Membranas Sinápticas/ultraestrutura , Transmissão Sináptica/fisiologia , Regulação para Cima/genéticaRESUMO
The expression and localization of the proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form acetylcholinesterase (G(4) AChE), were studied at vertebrate neuromuscular junctions. Both muscle and motor neuron contributed to this synaptic expression pattern. During the development of rat muscles, the expression of PRiMA and AChE(T) and the enzymatic activity increased dramatically; however, the proportion of G(4) AChE decreased. G(4) AChE in muscle was recognized specifically by a PRiMA antibody, indicating the association of this enzyme with PRiMA. Using western blot and ELISA, both PRiMA protein and PRiMA-linked G(4) AChE were found to be present in large amounts in fast-twitch muscle (e.g. tibialis), but in relatively low abundance in slow-twitch muscle (e.g. soleus). These results indicate that the expression level of PRiMA-linked G(4) AChE depends on muscle fiber type. In parallel, the expression of PRiMA, AChE(T) and G(4) AChE also increased in the spinal cord during development. Such expression in motor neurons contributed to the synaptic localization of G(4) AChE. After denervation, the expression of PRiMA, AChE(T) and G(4) AChE decreased markedly in the spinal cord, and in fast- and slow-twitch muscles.
Assuntos
Acetilcolinesterase/metabolismo , Proteínas de Membrana/metabolismo , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/metabolismo , Acetilcolinesterase/química , Processamento Alternativo , Animais , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas de Membrana/genética , Camundongos , Microscopia Confocal , Denervação Muscular , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismoRESUMO
The transcriptional regulation of proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form of acetylcholinesterase (G(4) AChE), was revealed in cultured cortical neurons during differentiation. The level of AChE(T) protein, total enzymatic activity and the amount of G(4) AChE were dramatically increased during the neuron differentiation. RT-PCR analyses revealed that the transcript encoding PRiMA was significantly up-regulated in the differentiated neurons. To investigate the transcriptional mechanism on PRiMA regulation, a reporter construct of human PRiMA promoter-tagged luciferase was employed in this study. Upon the neuronal differentiation in cortical neurons, a mitogen-activated protein (MAP) kinase-dependent pathway was stimulated: this signaling cascade was shown to regulate the transcriptional activity of PRiMA. In addition, both PRiMA and AChE(T) transcripts were induced by the over expression of an active mutant of Raf in the cultured neurons. The treatment of a MAP kinase inhibitor (U0126) significantly blocked the expression of PRiMA transcript and promoter-driven luciferase activity as induced by the differentiation of cortical neurons. These results suggested that a MAP kinase signaling pathway served as one of the transcriptional regulators in controlling PRiMA gene expression during the neuronal differentiation process.
Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neurogênese , Neurônios/fisiologia , Transcrição Gênica , Acetilcolinesterase/metabolismo , Animais , Butadienos/farmacologia , Linhagem Celular , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Nitrilas/farmacologia , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Quinases raf/genética , Quinases raf/metabolismoRESUMO
The collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase (AChE) in vertebrate muscles. Two ColQ transcripts, ColQ-1 and ColQ-1a, driven by two distinct promoters are expressed differentially in mammalian slow- and fast-twitch muscles, respectively. Such expression patterns are determined by the contractile activity in different muscle fiber types. To reveal the regulatory role of muscular activity on ColQ expression, acetylcholine and nicotine were applied onto C2C12 muscle cells: the challenge increased the expression of ColQ-1/ColQ-1a mRNAs. The agonist challenge induced the phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In parallel, over expression of an active mutant of CaMKII enhanced both ColQ-1/ColQ-1a mRNA levels in cultured C2C12 myotubes. Moreover, the over expression of myocyte enhancer factor 2 (MEF2), a downstream mediator of CaMKII, in the myotubes potentiated the CaMKII-induced ColQ expression. The current results reveal a signaling cascade that drives the expression profiles of ColQ in responding to activity challenge in cultured myotubes.
Assuntos
Acetilcolina/metabolismo , Acetilcolinesterase/metabolismo , Colágeno/metabolismo , Regulação da Expressão Gênica , Fibras Musculares Esqueléticas/fisiologia , Proteínas Musculares/metabolismo , Fatores de Regulação Miogênica/metabolismo , Acetilcolinesterase/genética , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Colágeno/genética , Humanos , Fatores de Transcrição MEF2 , Camundongos , Fibras Musculares Esqueléticas/citologia , Proteínas Musculares/genética , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Fatores de Regulação Miogênica/genética , Regiões Promotoras Genéticas , Ratos , Receptores Colinérgicos/metabolismoRESUMO
Acetylcholinesterase (AChE) is a highly polymorphic enzyme. Alternative splicing in the 3' region of the primary transcript generates different subunits that contain the same catalytic domain but with distinct carboxyl termini. In mammals, the AChE(R) variant produces a soluble monomer that is up-regulated in the brain during stress. The AChE(H) variant produces a GPI-anchored dimer that is mainly expressed in blood cells, while AChE(T) variant is largely predominant in the brain and muscle. AChE(T) subunits associate with a collagen tail subunit (ColQ) forming asymmetric AChE species (A(4), A(8), and A(12) AChE) in muscle, and also form amphiphilic tetramers associated with a proline-rich membrane anchor (PRiMA) as globular AChE (G(4) AChE) in brain and muscle. The formation of these AChE forms depends on the physiological status of the muscles, and on the innervating nerves. The motor nerves achieve this regulation by two distinct mechanisms: release of the trophic factor calcitonin gene-related peptide (CGRP) and nerve-evoked electrical activity, which differentially regulate the expression levels of AChE(T), PRiMA and ColQ via different downstream signaling cascades. The regulatory mechanisms provided by the nerve are important to account for the different expression patterns of AChE and associated proteins in fast- and slow-twitch muscles.
Assuntos
Acetilcolinesterase/genética , Neurônios Motores/metabolismo , Músculos/enzimologia , Transdução de Sinais , Transcrição Gênica , Animais , Peptídeo Relacionado com Gene de Calcitonina/fisiologia , Diferenciação Celular , Perfilação da Expressão Gênica , Humanos , Músculos/citologia , Nervo Isquiático/fisiologiaRESUMO
The tetrameric globular form of acetylcholinesterase (G(4) AChE) is present and precisely controlled in muscles. The assembly and membrane targeting of G(4) AChE are directed by a proline-rich membrane anchor (PRiMA). It has been demonstrated that in muscle cells, the expression of PRiMA mRNA, as well as the level of G(4) AChE was suppressed by myogenesis and innervating nerve. A human PRiMA promoter-driven luciferase reporter was employed in this study to further reveal the activity of PRiMA transcription during myogenic differentiation and the influence of innervation. In parallel with PRiMA mRNA, the PRiMA promoter activity was suppressed by both myogenic regulatory factor(s) (MRFs) and nerve-derived factor(s). These results suggest that the regulation of PRiMA mRNA expression in muscle by MRFs and nerve-derived factors is due to a control system at the transcriptional level.
Assuntos
Acetilcolinesterase/genética , Proteínas de Membrana/genética , Músculos/enzimologia , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Sequência de Bases , Primers do DNA , Humanos , Proteínas de Membrana/metabolismo , Músculos/inervação , Proteínas do Tecido Nervoso/metabolismo , Conformação Proteica , RNA Mensageiro/genéticaRESUMO
The presence of a collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase at vertebrate neuromuscular junctions (nmjs). Two ColQ transcripts as ColQ-1 and ColQ-1a, driven by two promoters: pColQ-1 and pColQ-1a, were found in mammalian slow- and fast-twitch muscles, respectively, which have distinct expression pattern in different muscle fibers. In this study, we show the differential expression of CoQ in different muscles is triggered by calcitonin gene-related peptide (CGRP), a known motor neuron-derived factor. Application of CGRP, or dibutyryl-cAMP (Bt(2)-cAMP), in cultured myotubes induced the expression of ColQ-1a transcript and promoter activity; however, the expression of ColQ-1 transcript did not respond to CGRP or Bt(2)-cAMP. The CGRP-induced gene activation was blocked by an adenylyl cyclase inhibitor or a dominant negative mutant of cAMP-responsive element (CRE) binding protein (CREB). Two CRE sites were mapped within the ColQ-1a promoter, and mutations of the CRE sites abolished the response of CGRP or Bt(2)-cAMP. In parallel, CGRP receptor complex was dominantly expressed at the nmjs of fast muscle but not of slow muscle. These results suggested that the expression of ColQ-1a at the nmjs of fast-twitch muscle was governed by a CGRP-mediated cAMP signaling mechanism.
Assuntos
Acetilcolinesterase/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Células Musculares/citologia , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fibras Musculares de Contração Lenta/efeitos dos fármacos , Animais , Bucladesina/farmacologia , Proteína de Ligação a CREB/metabolismo , Células Cultivadas , Embrião de Galinha , Galinhas , Imunoprecipitação da Cromatina/métodos , Relação Dose-Resposta a Droga , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Receptores Colinérgicos/metabolismo , Fatores de Tempo , Ativação Transcricional , Transfecção/métodosRESUMO
The transcriptional regulation of proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form acetylcholinesterase (G(4) AChE), was revealed in muscle during myogenic differentiation under the influence of innervation. During myotube formation of C2C12 cells, the expression of AChE(T) protein and the enzymatic activity were dramatically increased, but the level of G(4) AChE was relatively decreased. This G(4) AChE in C2C12 cells was specifically recognized by anti-PRiMA antibody, suggesting the association of this enzyme with PRiMA. Reverse transcription-PCR analysis revealed that the level of PRiMA mRNA was reduced during the myogenic differentiation of C2C12 cells. Overexpression of PRiMA in C2C12 myotubes significantly increased the production of G(4) AChE. The oligomerization of G(4) AChE, however, did not require the intracellular cytoplasmic tail of PRiMA. After overexpressing the muscle regulatory factors, myogenin and MyoD, the expressions of PRiMA and G(4) AChE in cultured myotubes were markedly reduced. In addition, calcitonin gene-related peptide, a known motor neuron-derived factor, and muscular activity were able to suppress PRiMA expression in muscle; the suppression was mediated by the phosphorylation of a cAMP-responsive element-binding protein. In accordance with the in vitro results, sciatic nerve denervation transiently increased the expression of PRiMA mRNA and decreased the phosphorylation of cAMP-responsive element-binding protein as well as its activator calcium/calmodulin-dependent protein kinase II in muscles. Our results suggest that the expression of PRiMA, as well as PRiMA-associated G(4) AChE, in muscle is suppressed by muscle regulatory factors, muscular activity, and nerve-derived trophic factor(s).