RESUMO
Experimental studies have demonstrated that methylphenidate (MPH) modulates the synaptic vesicle trafficking and synaptotagmin-1 (SytI) mRNA levels. SytI is a regulatory protein of the SNARE complex, a neurotransmitter exocytosis mediator. Despite this evidence, most SNARE complex-related genes have never been evaluated in attention-deficit/hyperactivity disorder (ADHD) pharmacogenetics. This study evaluates, for we believe the first time, polymorphisms on the SNARE complex-related genes STX1A (rs2228607), VAMP2 (26bp Ins/Del) and SYT1 (rs1880867 and rs2251214) on the response to immediate-release methylphenidate (IR-MPH) in a naturalistic sample of adults with ADHD. The sample comprised 433 subjects, of which 272 (62.8%) have completed the short-term IR-MPH treatment (at least 30 days). The main outcome measure was the categorical variable of short-term response to IR-MPH based on the Swanson, Nolan and Pelham Rating Scale version 4 (SNAP-IV), and on the clinical global impression-improvement scale. Additional analyses evaluated the percentage of SNAP-IV symptom reduction for each dimension as well as short- and long- (7 years) term treatment persistence. SYT1-rs2251214 was associated with the categorical short-term response to IR-MPH (P=0.006, PFDR=0.028), and with the percentage of inattention and oppositional defiant disorder symptoms reduction (P=0.007, PFDR=0.028 and P=0.017, PFDR=0.048, respectively). SYT1-rs2251214 was also associated with short-term treatment persistence (P=0.018, PFDR=0.048), and with months of treatment (P=0.002, PFDR=0.016) in the long-term protocol. Our findings suggest that SYT1-rs2251214 presents a broad influence in IR-MPH response variability in adults with ADHD, being involved with both symptom response and treatment persistence. If such findings are replicated, SytI could represent a key element in MPH pharmacodynamics in adults with ADHD.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Exocitose/genética , Sinaptotagmina I/genética , Adulto , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Transtornos de Deficit da Atenção e do Comportamento Disruptivo/complicações , Estimulantes do Sistema Nervoso Central , Exocitose/fisiologia , Feminino , Humanos , Masculino , Metilfenidato/farmacologia , Metilfenidato/uso terapêutico , Avaliação de Resultados em Cuidados de Saúde , Polimorfismo Genético , Sinaptotagmina I/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismo , Resultado do Tratamento , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
The organophosphate and carbamate pesticides methyl-parathion and carbaryl have a common action mechanism: they inhibit acetylcholinesterase enzyme by blocking the transmission of nerve impulses. However, they can alter the expression of exocytotic membrane proteins (SNARE), by modifying release of neurotransmitters and other substances. This study evaluated the adverse effects of the pesticides methyl-parathion and carbaryl on expression of SNARE proteins: Syntaxin-1, Syntaxin-4 and SNAP-23 in freshwater rotifer Brachionus calyciflorus. Protein expression of these three proteins was analyzed before and after exposure to these two pesticides by Western Blot. The expression of Syntaxin-1, Syntaxin-4 and SNAP-23 proteins in B. calyciflorussignificantly decreases with increasing concentration of either pesticides. This suggests that organophosphates and carbamates have adverse effects on expression of membrane proteins of exocytosis by altering the recognition, docking and fusion of presynaptic and vesicular membranes involved in exocytosis of neurotransmitters. Our results demonstrate that the neurotoxic effect of anticholinesterase pesticides influences the interaction of syntaxins and SNAP-25 and the proper assembly of the SNARE complex.
Assuntos
Carbaril/farmacologia , Inseticidas/farmacologia , Metil Paration/farmacologia , Rotíferos/efeitos dos fármacos , Animais , Inibidores da Colinesterase/farmacologia , Proteínas Qa-SNARE/metabolismo , Rotíferos/enzimologia , Sintaxina 1/metabolismoRESUMO
AbstractThe organophosphate and carbamate pesticides methyl-parathion and carbaryl have a common action mechanism: they inhibit acetylcholinesterase enzyme by blocking the transmission of nerve impulses. However, they can alter the expression of exocytotic membrane proteins (SNARE), by modifying release of neurotransmitters and other substances. This study evaluated the adverse effects of the pesticides methyl-parathion and carbaryl on expression of SNARE proteins: Syntaxin-1, Syntaxin-4 and SNAP-23 in freshwater rotifer Brachionus calyciflorus. Protein expression of these three proteins was analyzed before and after exposure to these two pesticides by Western Blot. The expression of Syntaxin-1, Syntaxin-4 and SNAP-23 proteins in B. calyciflorussignificantly decreases with increasing concentration of either pesticides. This suggests that organophosphates and carbamates have adverse effects on expression of membrane proteins of exocytosis by altering the recognition, docking and fusion of presynaptic and vesicular membranes involved in exocytosis of neurotransmitters. Our results demonstrate that the neurotoxic effect of anticholinesterase pesticides influences the interaction of syntaxins and SNAP-25 and the proper assembly of the SNARE complex.
ResumoOs pesticidas organofosforados e carbamatos metil- paration e carbaril tem um mecanismo de ação comum: eles inibem a enzima acetilcolinesterase, bloqueando a transmissão dos impulsos nervosos. No entanto, eles podem alterar a expressão de proteínas de membrana de exocitose (SNARE), através da modificação da libertação de neurotransmissores e outras substâncias. Este estudo avaliou os efeitos adversos dos pesticidas metil- paration e carbaril sobre a expressão de proteínas SNARE: Sintaxina -1, Sintaxina-4 e SNAP-23 em rotíferos de água doce Brachionus calyciflorus. A expressão destas três proteínas foi analisada antes e depois da exposição a estes dois pesticidas por Western Blot. A expressão das proteínas Sintaxina-1, Sintaxina-4 e SNAP-23 em B. calyciflorus diminui significativamente com o aumento da concentração de ambos os pesticidas. Isto sugere que os organofosfatos e carbamatos têm efeitos adversos sobre a expressão de proteínas de membrana de exocitose, alterando o reconhecimento, de encaixe e fusão de membranas pré-sinápticas e vesiculares envolvidas na exocitose de neurotransmissores. Nossos resultados demonstram que o efeito neurotóxico de pesticidas anticolinesterásicos influencia a interação de sintaxinas e SNAP-25 e a montagem correta do complexo SNARE.
Assuntos
Animais , Carbaril/farmacologia , Inseticidas/farmacologia , Metil Paration/farmacologia , Rotíferos/efeitos dos fármacos , Inibidores da Colinesterase/farmacologia , Proteínas Qa-SNARE/metabolismo , Rotíferos/enzimologia , Sintaxina 1/metabolismoRESUMO
The organophosphate and carbamate pesticides methyl-parathion and carbaryl have a common action mechanism: they inhibit acetylcholinesterase enzyme by blocking the transmission of nerve impulses. However, they can alter the expression of exocytotic membrane proteins (SNARE), by modifying release of neurotransmitters and other substances. This study evaluated the adverse effects of the pesticides methyl-parathion and carbaryl on expression of SNARE proteins: Syntaxin-1, Syntaxin-4 and SNAP-23 in freshwater rotifer Brachionus calyciflorus. Protein expression of these three proteins was analyzed before and after exposure to these two pesticides by Western Blot. The expression of Syntaxin-1, Syntaxin-4 and SNAP-23 proteins in B. calyciflorussignificantly decreases with increasing concentration of either pesticides. This suggests that organophosphates and carbamates have adverse effects on expression of membrane proteins of exocytosis by altering the recognition, docking and fusion of presynaptic and vesicular membranes involved in exocytosis of neurotransmitters. Our results demonstrate that the neurotoxic effect of anticholinesterase pesticides influences the interaction of syntaxins and SNAP-25 and the proper assembly of the SNARE complex.(AU)
Os pesticidas organofosforados e carbamatos metil- paration e carbaril tem um mecanismo de ação comum: eles inibem a enzima acetilcolinesterase, bloqueando a transmissão dos impulsos nervosos. No entanto, eles podem alterar a expressão de proteínas de membrana de exocitose (SNARE), através da modificação da libertação de neurotransmissores e outras substâncias. Este estudo avaliou os efeitos adversos dos pesticidas metil- paration e carbaril sobre a expressão de proteínas SNARE: Sintaxina -1, Sintaxina-4 e SNAP-23 em rotíferos de água doce Brachionus calyciflorus. A expressão destas três proteínas foi analisada antes e depois da exposição a estes dois pesticidas por Western Blot. A expressão das proteínas Sintaxina-1, Sintaxina-4 e SNAP-23 em B. calyciflorus diminui significativamente com o aumento da concentração de ambos os pesticidas. Isto sugere que os organofosfatos e carbamatos têm efeitos adversos sobre a expressão de proteínas de membrana de exocitose, alterando o reconhecimento, de encaixe e fusão de membranas pré-sinápticas e vesiculares envolvidas na exocitose de neurotransmissores. Nossos resultados demonstram que o efeito neurotóxico de pesticidas anticolinesterásicos influencia a interação de sintaxinas e SNAP-25 e a montagem correta do complexo SNARE.(AU)
Assuntos
Animais , Carbaril/farmacologia , Inseticidas/farmacologia , Metil Paration/farmacologia , Rotíferos , Inibidores da Colinesterase/farmacologia , Proteínas Qa-SNARE/metabolismo , Rotíferos/enzimologia , Sintaxina 1/metabolismoRESUMO
Syntaxin-1 and 25-kDa Synaptosome-associated Protein (SNAP-25) are present in the plasma membrane of several different secretory cell types and are involved in the exocytosis process. In this work, the free-living amoeba Difflugia corona was studied in relation to ultrastructure, structural membrane proteins, and proteins such as Syntaxin-1 and SNAP-25. Our results obtained by scanning electron microscopy in the amoeba without its theca, showed many membrane projections and several pore-like structures. Using immunocytochemistry, we found structural proteins Syntaxin-1 and SNAP-25.
Assuntos
Animais , Amoeba/metabolismo , Amoeba/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , /metabolismo , Proteínas de Membrana/metabolismo , Sintaxina 1/metabolismo , Microscopia Eletrônica de VarreduraRESUMO
Syntaxin-1 and 25-kDa Synaptosome-associated Protein (SNAP-25) are present in the plasma membrane of several different secretory cell types and are involved in the exocytosis process. In this work, the free-living amoeba Difflugia corona was studied in relation to ultrastructure, structural membrane proteins, and proteins such as Syntaxin-1 and SNAP-25. Our results obtained by scanning electron microscopy in the amoeba without its theca, showed many membrane projections and several pore-like structures. Using immunocytochemistry, we found structural proteins Syntaxin-1 and SNAP-25.(AU)
Assuntos
Animais , Amoeba/metabolismo , Amoeba/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas de Membrana/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Microscopia Eletrônica de VarreduraRESUMO
Prolactin induces maturation of insulin secretion in cultured neonatal rat islets. In this study, we investigated whether the improved secretory response to glucose caused by prolactin involves alteration in the expression, association and phosphorylation of several proteins that participate in these processes. Messenger RNA was extracted from neonatal rat islets cultured for 5 days in the presence of prolactin and reverse transcribed. Gene expression was analyzed by semi-quantitative RT-PCR and by Western blotting for proteins. The gene transcription and protein expression of kinesin and MAP-2 were increased in prolactin-treated islets compared to the controls. The association and phosphorylation of proteins was analyzed by immunoprecipitation followed by Western blotting, after acute exposure to prolactin. Prolactin increased the association between SNARE proteins and kinesin/MAP-2 while the association of munc-18/syntaxin 1A was decreased. Serine phosphorylation of SNAP-25, syntaxin 1A, munc-18, MAP-2 was significantly higher whereas kinesin phosphorylation was decreased in prolactin-treated islets. There was an increase in SNARE complex formation in islets stimulated with prolactin, 22 mM glucose, 40 mM K(+), 200 microM carbachol and 1 microM PMA. The prolactin-induced increase in the formation of SNARE complex and syntaxin 1A phosphorylation was inhibited by PD098059 and U0126, inhibitors of the MAPK pathway. These findings indicate that prolactin primes pancreatic beta-cells to release insulin by increasing the expression and phosphorylation/association of proteins implicated in the secretory machinery and the MAPK/PKC pathway is important for this effect.
Assuntos
Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Prolactina/farmacologia , Proteínas SNARE/metabolismo , Animais , Animais Recém-Nascidos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/enzimologia , Cinesinas/genética , Potenciais da Membrana/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Fatores de TempoRESUMO
We assessed immunoreactivity (IR) in the cerebral cortex (CC), hippocampus (Hipp), and striatum (ST) of a growth-associated protein, GAP-43, and of proteins of the synaptic vesicle fusion complex: VAMP-2, Syntaxin-1, and SNAP-25 (SNARE proteins) throughout postnatal development of rats after submitting the animals to acute global postnatal hypoxia (6.5% O(2), 70 min) at postnatal day 4 (PND4). In the CC only the IR of the SNARE protein SNAP-25 increased significantly with age. The hypoxic animals showed the same pattern of IR for SNAP-25, although with lower levels at PND11, and also a significant increase of VAMP-2. SNAP-25 (control): PND11 P < 0.001 vs. PND18, 25, and 40, SNAP-25 (hypoxic): P < 0.001 vs. PND18, 25, and 40; VAMP-2 (hypoxic): P < 0.05 PND11 vs. PND18, and P < 0.01 vs. PND25 and PND40; one-way ANOVA and Bonferroni post-test. In the Hipp, SNAP-25 and syntaxin-1 increased significantly with age, reaching a plateau at PND25 through PND40 in control animals (one-way ANOVA: syntaxin-1: P = 0.043; Bonferroni: NS; SNAP-25: P = 0.013; Bonferroni: P < 0.01 PND11 vs. PND40). Hypoxic rats showed higher levels of significance in the one-way ANOVA than controls (syntaxin-1: P = 0.009; Bonferroni: P < 0.05 PND11 vs. PND25 and P < 0.001 PND11 vs. PND40). In the ST, GAP-43 differed significantly among hypoxic and control animals and the two-way ANOVA revealed significant differences with age (F = 3.23; P = 0.037) and treatment (F = 4.84; P = 0.036). VAMP-2 expression also reached statistical significance when comparing control and treated animals (F = 6.25, P = 0.018) without changes regarding to age. Elevated plus maze test performed at PND40 indicated a lower level of anxiety in the hypoxic animals. At adulthood (12 weeks) learning, memory and locomotor abilities were identical in both groups of animals. With these results, we demonstrate that proteins of the presynaptic structures of the ST are sensitive to acute disruption of homeostatic conditions, such as a temporary decrease of the O(2) concentration. Modifications in the activity of these proteins could contribute to the long term altered responses to stress due to acute hypoxic insult in the neonatal period.
Assuntos
Corpo Estriado/metabolismo , Proteína GAP-43/metabolismo , Hipóxia Encefálica/metabolismo , Regulação para Cima/fisiologia , Doença Aguda , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Transtornos de Ansiedade/etiologia , Transtornos de Ansiedade/metabolismo , Transtornos de Ansiedade/fisiopatologia , Câmaras de Exposição Atmosférica , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Corpo Estriado/fisiopatologia , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Hipóxia Encefálica/fisiopatologia , Imuno-Histoquímica , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Ratos , Ratos Sprague-Dawley , Estresse Fisiológico/metabolismo , Estresse Fisiológico/fisiopatologia , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
During pregnancy and the perinatal period of life, prolactin (PRL) and other lactogenic substances induce adaptation and maturation of the stimulus-secretion coupling system in pancreatic beta-cells. Since the SNARE molecules, SNAP-25, syntaxin 1, VAMP-2, and synaptotagmins participate in insulin secretion, we investigated whether the improved secretory response to glucose during these periods involves alteration in the expression of these proteins. mRNA was extracted from neonatal rat islets cultured for 5 days in the presence of PRL and from pregnant rats (17th-18th days of pregnancy) and reverse transcribed. The expression of genes was analyzed by semi-quantitative RT-PCR assay. The expression of proteins was analyzed by Western blotting and confocal microscopy. Transcription and expression of all SNARE genes and proteins were increased in islets from pregnant and PRL-treated neonatal rats when compared with controls. The only exception was VAMP-2 production in islets from pregnant rats. Increased mRNA and protein expression of synaptotagmin IV, but not the isoform I, also was observed in islets from pregnant and PRL-treated rats. This effect was not inhibited by wortmannin or PD098059, inhibitors of the PI3-kinase and MAPK pathways, respectively. As revealed by confocal laser microscopy, both syntaxin 1A and synaptotagmin IV were immunolocated in islet cells, including the insulin-containing cells. These results indicate that PRL modulates the final steps of insulin secretion by increasing the expression of proteins involved in membrane fusion.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Prolactina/farmacologia , Proteínas SNARE/genética , Sinaptotagminas/genética , Animais , Animais Recém-Nascidos , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Immunoblotting , Imunoquímica , Insulina/genética , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/embriologia , Microscopia Confocal , Gravidez , RNA Mensageiro/análise , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptotagminas/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
During pregnancy and the perinatal period of life, prolactin (PRL) and other lactogenic substances induce adaptation and maturation of the stimulus-secretion coupling system in pancreatic â-cells. Since the SNARE molecules, SNAP-25, syntaxin 1, VAMP-2, and synaptotagmins participate in insulin secretion, we investigated whether the improved secretory response to glucose during these periods involves alteration in the expression of these proteins. mRNA was extracted from neonatal rat islets cultured for 5 days in the presence of PRL and from pregnant rats (17th-18th days of pregnancy) and reverse transcribed. The expression of genes was analyzed by semi-quantitative RT-PCR assay. The expression of proteins was analyzed by Western blotting and confocal microscopy. Transcription and expression of all SNARE genes and proteins were increased in islets from pregnant and PRL-treated neonatal rats when compared with controls. The only exception was VAMP-2 production in islets from pregnant rats. Increased mRNA and protein expression of synaptotagmin IV, but not the isoform I, also was observed in islets from pregnant and PRL-treated rats. This effect was not inhibited by wortmannin or PD098059, inhibitors of the PI3-kinase and MAPK pathways, respectively. As revealed by confocal laser microscopy, both syntaxin 1A and synaptotagmin IV were immunolocated in islet cells, including the insulin-containing cells. These results indicate that PRL modulates the final steps of insulin secretion by increasing the expression of proteins involved in membrane fusion.