RESUMO
AIMS: This work explored the effects of irisin on metabolism, gene expression and mitochondrial content in cultured myocytes. METHODS: C2C12 myocytes were treated with various concentrations of irisin for various durations. Glycolysis and oxidative metabolism were quantified by measurement of extracellular acidification and oxygen consumption, respectively. Metabolic gene expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and mitochondrial content was assessed by flow cytometry and confocal microscopy. RESULTS: Cells treated with irisin exhibited significantly increased oxidative metabolism. Irisin treatment also significantly increased mitochondrial uncoupling at various doses and durations. Lastly, treatment with irisin also significantly elevated metabolic gene expression including peroxisome proliferator-activated receptor γ coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), irisin, glucose transporter 4 (GLUT4) and mitochondrial uncoupling protein 3 (UCP3) leading to increased mitochondrial biogenesis. CONCLUSIONS: Our observations are the first to document increased metabolism in myocytes through irisin-mediated induction of mitochondrial biogenesis and uncoupling with corresponding gene expression. These observations support the need for further investigation into the therapeutic and pharmacological effects of irisin, as well as development of irisin-based therapy.
Assuntos
Fibronectinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Mitocôndrias Musculares/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Proteínas Musculares/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibronectinas/agonistas , Fibronectinas/genética , Fibronectinas/metabolismo , Proteínas de Grupo de Alta Mobilidade/agonistas , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Cinética , Camundongos , Mitocôndrias Musculares/metabolismo , Renovação Mitocondrial/efeitos dos fármacos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/agonistas , Proteínas Musculares/genética , Fator 1 Nuclear Respiratório/agonistas , Fator 1 Nuclear Respiratório/genética , Fator 1 Nuclear Respiratório/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas Recombinantes/farmacologia , Fatores de Transcrição/agonistas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The dopamine transporter (DAT) is a neuronal phosphoprotein and target for psychoactive drugs that plays a critical role in terminating dopaminergic transmission by reuptake of dopamine from the synaptic space. Control of DAT activity and plasma membrane expression are therefore central to drug actions and the spatial and temporal regulation of synaptic dopamine levels. DATs rapidly traffic between the plasma membrane and endosomal compartments in both constitutive and protein kinase C-dependent manners. Kinase activators, phosphatase inhibitors, and transported substrates modulate DAT phosphorylation and activity, but the underlying mechanisms and role of phosphorylation in these processes are poorly understood. Complex adaptive changes in DAT function potentially related to these processes are also induced by psychostimulant and therapeutic transport blockers such as cocaine and methylphenidate. This chapter provides an overview of the current state of knowledge regarding DAT phosphorylation and its relationship to transporter activity and trafficking. A better understanding of how dopaminergic neurons regulate DAT function and the role of phosphorylation may lead to the identification of novel therapeutic targets for the treatment and prevention of dopaminergic disorders.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Processamento de Proteína Pós-Traducional , Anfetamina/farmacologia , Animais , Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/farmacologia , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Inibidores da Captação de Dopamina/farmacologia , Humanos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Conformação Proteica , Transporte ProteicoRESUMO
Coadministration of kappa-opioid receptor agonists (kappa-agonists) with cocaine prevents alterations in dialysate dopamine (DA) concentration in the nucleus accumbens (Acb) that occur during abstinence from repeated cocaine treatment. Quantitative microdialysis was used to determine the mechanism producing these effects. Rats were injected with cocaine (20 mg/kg, i.p.), or saline, and the selective kappa-agonist U-69593 (0.32 mg/kg, s.c.), or vehicle, once daily for 5 d. Extracellular DA concentration (DA(ext)) and extraction fraction (E(d)), an indirect measure of DA uptake, were determined 3 d later. Repeated cocaine treatment increased E(d), whereas repeated U-69593 treatment decreased E(d), relative to controls. Coadministration of both drugs yielded intermediate E(d) values not different from controls. In vitro DA uptake assays confirmed that repeated U-69593 treatment produces a dose-related, region-specific decrease in DA uptake and showed that acute U-69593 administration increases DA uptake in a nor-binaltorphimine reversible manner. Repeated U-69593 also led to a decrease in [(125)I]RTI-55 binding to the DA transporter (DAT), but did not decrease total DAT protein. These results demonstrate that kappa-opioid receptor activation modulates DA uptake in the Acb in a manner opposite to that of cocaine: repeated U-69593 administration decreases the basal rate of DA uptake, and acute U-69593 administration transiently increases DA uptake. kappa-agonist treatment also alters DAT function. The action of kappa-agonists on DA uptake or DAT binding, or both, may be the mechanism(s) mediating the previously reported "cocaine-antagonist" effect of kappa-opioid receptor agonists.
Assuntos
Benzenoacetamidas , Cocaína/antagonistas & inibidores , Dopamina/farmacocinética , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Naltrexona/análogos & derivados , Proteínas do Tecido Nervoso , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Receptores Opioides kappa/metabolismo , Análise de Variância , Animais , Autorradiografia , Proteínas de Transporte/metabolismo , Cocaína/administração & dosagem , Corpo Estriado/química , Corpo Estriado/metabolismo , Dopamina/análise , Proteínas da Membrana Plasmática de Transporte de Dopamina , Esquema de Medicação , Antagonismo de Drogas , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Injeções Intraperitoneais , Injeções Subcutâneas , Radioisótopos do Iodo , Modelos Lineares , Masculino , Microdiálise , Naltrexona/administração & dosagem , Núcleo Accumbens/química , Pirrolidinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Receptores Opioides kappa/agonistasRESUMO
Presynaptic transporter proteins regulate the clearance of extracellular biogenic amines after release and are important targets for multiple psychoactive agents, including amphetamines, cocaine, and antidepressant drugs. Recent studies reveal that dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters (DAT, NET, and SERT, respectively) are rapidly regulated by direct or receptor-mediated activation of cellular kinases, particularly protein kinase C (PKC). With SERTs, PKC activation results in activity-dependent transporter phosphorylation and sequestration. Protein phosphatase 1/2A (PP1/PP2A) inhibitors, such as okadaic acid (OA) and calyculin A, also promote SERT phosphorylation and functional downregulation. How kinase, phosphatase, and transporter activities are linked mechanistically is unclear. In the present study, we found that okadaic acid-sensitive phosphatase activity is enriched in SERT immunoprecipitates from human SERT stably transfected cells. Moreover, blots of these immunoprecipitates reveal the presence of PP2A catalytic subunit (PP2Ac), findings replicated using brain preparations. Whole-cell treatments with okadaic acid or calyculin A diminished SERT/PP2Ac associations. Phorbol esters, which trigger SERT phosphorylation, also diminish SERT/PP2Ac associations, effects that can be blocked by PKC antagonists as well as the SERT substrate 5-HT. Similar transporter/PP2Ac complexes were also observed in coimmunoprecipitation studies with NETs and DATs. Our findings provide evidence for the existence of regulated heteromeric assemblies involving biogenic amine transporters and PP2A and suggest that the dynamic stability of these complexes may govern transporter phosphorylation and sequestration.
Assuntos
Antidepressivos/farmacologia , Aminas Biogênicas/metabolismo , Cocaína/farmacologia , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Fosfoproteínas Fosfatases/metabolismo , Simportadores , Animais , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas da Membrana Plasmática de Transporte de Dopamina , Humanos , Substâncias Macromoleculares , Toxinas Marinhas , Glicoproteínas de Membrana/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina , Ácido Okadáico/farmacologia , Oxazóis/farmacologia , Ésteres de Forbol/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Proteína Quinase C/antagonistas & inibidores , Proteína Fosfatase 1 , Proteína Fosfatase 2 , Transporte Proteico/efeitos dos fármacos , Serotonina/metabolismo , Serotonina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina , TransfecçãoRESUMO
Cholinergic activation of dopaminergic neurons in the ventral tegmental area (VTA) is thought to play a major role in cognitive functions and reward. These dopaminergic neurons differentially project to cortical and limbic forebrain regions, where their terminals differ in levels of expression of the plasmalemmal dopamine transporter (DAT). This transporter selectively identifies dopaminergic neurons, whereas the vesicular acetylcholine transporter (VAchT) is present only in the neurons that store and release acetylcholine. We examined immunogold labeling for DAT and immunoperoxidase localization of VAchT antipeptide antisera in single sections of the rat VTA to determine whether dopaminergic somata and dendrites in this region differ in their levels of expression of DAT and/or input from cholinergic terminals. VAchT immunoreactivity was prominently localized to membranes of small synaptic vesicles in unmyelinated axons and axon terminals. VAchT-immunoreactive terminals formed almost exclusively asymmetric synapses with dendrites. Of 159 dendrites that were identified as cholinergic targets, 35% contained plasmalemmal DAT, and 65% were without detectable DAT immunoreactivity. The DAT-immunoreactive dendrites postsynaptic to VAchT-labeled terminals contained less than half the density of gold particles as seen in other dendrites receiving input only from unlabeled terminals. These results suggest selective targeting of cholinergic afferents in the VTA to non-dopaminergic neurons and a subpopulation of dopaminergic neurons that have a limited capacity for plasmalemmal reuptake of dopamine, a characteristic of those that project to the frontal cortex.
Assuntos
Acetilcolina/metabolismo , Proteínas de Transporte/metabolismo , Dopamina/metabolismo , Terminações Nervosas/metabolismo , Neurônios Aferentes/metabolismo , Terminações Pré-Sinápticas/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Cognição/fisiologia , Técnicas de Cultura , Dendritos/metabolismo , Sistema Límbico/metabolismo , Masculino , Microscopia Eletrônica , Terminações Nervosas/citologia , Neurônios Aferentes/citologia , Prosencéfalo/metabolismo , Ratos , Ratos Sprague-Dawley , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Área Tegmentar Ventral/citologiaRESUMO
The dopamine transporter (DAT) is a primary site for the action of cocaine in inducing euphoria. Its action is necessary for the selectivities of dopaminergic neurotoxins that provide the best current experimental models of Parkinson's disease. In the present report, rat dopamine transporter-like immunoreactivity (iDAT) was assessed by immunohistochemistry using newly developed polyclonal antisera raised against conjugated peptides corresponding to sequences found in the dopamine transporter's carboxy- and amino-termini. Dense iDAT was observed in patterns consistent with neural processes and terminals in the striatum, nucleus accumbens, olfactory tubercle, nigrostriatal bundle, and lateral habenula. Perikarya in the substantia nigra pars compacta were immunostained with moderate intensity using one of two immunohistochemical methods, while scattered ventral tegmental area perikarya were stained with somewhat less intensity. Immunoreactive neuronal processes with axonal and dendritic morphologies were stained in the substantia nigra and the paranigral and parabrachialis pigmentosus nuclei of the ventral tegmental area, while sparser processes were noted more medially in the ventral tegmental area. Neuronal processes were found in several laminae in the cingulate cortex, with notable fiber densities in the superficial aspects of lamina I and laminae II/III. The intensities of immunoreactivities in striatum and cerebral cortex were dramatically attenuated ipsilateral to nigrostriatal bundle 6-hydroxydopamine lesions. Specificity of immunostaining was supported by agreement of the results using sera directed against two distinct DAT segments, studies with preimmune and preadsorbed sera and studies of the extracted protein. These antisera identify and reveal details of the distribution of DAT immunoreactivity in rat brain and display variations in levels of DAT expression of likely functional significance.
Assuntos
Química Encefálica/fisiologia , Proteínas de Transporte/análise , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso/análise , Neuropeptídeos/análise , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Proteínas da Membrana Plasmática de Transporte de Dopamina , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Ratos , Ratos Sprague-DawleyRESUMO
Several aspects of DAT structure and function have been elucidated using a combination of photoaffinity labeling, proteolysis, enzymatic deglycosylation, and epitope-specific immunoprecipitation. The two photolabels are incorporated in different regions of the protein, suggesting that the binding sites for the ligands are distinct or partially nonoverlapping, consistent with results produced by site-directed mutagenesis and analysis of chimeras. These studies have also verified several aspects of DAT structure previously hypothesized based only on theoretical considerations, including the presence of at least one transmembrane helix or other membrane-anchoring structure in two different regions of the protein, identification of the glycosylated domain, and some topological properties. It should be possible to extend and adapt these techniques to further delineate DAT structural properties and to identify other functional domains such as phosphorylation sites or active sulfhydryl moieties.
Assuntos
Marcadores de Afinidade , Azidas/química , Encéfalo/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cocaína/análogos & derivados , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Piperazinas/química , Animais , Azidas/farmacocinética , Sítios de Ligação , Carboidratos/análise , Proteínas de Transporte/isolamento & purificação , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , Cocaína/química , Cocaína/farmacocinética , Proteínas da Membrana Plasmática de Transporte de Dopamina , Glicosilação , Humanos , Radioisótopos do Iodo , Mutagênese Sítio-Dirigida , Piperazinas/farmacocinética , Ensaio Radioligante/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The cocaine analog [3H]WIN 35,428 was used to label digitonin-solubilized dopamine transporters from dog caudate nucleus. The assay consists of incubation of extracts with the ligand followed by separation of free from bound ligand by centrifugation after adding activated charcoal. Specific binding was observed in dog caudate but was absent in dog cerebellar extracts. Binding was linear with tissue, saturable, and of high affinity (Kd = 16 nM). In competition studies, soluble [3H]WIN 35,428 binding was inhibited strongly by mazindol, GBR 12909, and (-)-cocaine but only weakly by citalopram, desipramine, and (+)-cocaine; this is typical of binding to the dopamine transporter. Compared to assays using [3H]GBR 12935, (-)-cocaine was relatively more potent, suggesting that the cocaine and GBR 12935 binding sites are somewhat different. When soluble extract was chromatographed on a wheat germ agglutinin-Sepharose column, [3H]WIN 35,428 binding activity was eluted with N-acetylglucosamine in a manner similar to photoaffinity-labeled dopamine transporters.
Assuntos
Proteínas de Transporte/análise , Cocaína/análogos & derivados , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Adsorção , Animais , Ligação Competitiva/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Proteínas de Transporte/farmacocinética , Núcleo Caudado/efeitos dos fármacos , Núcleo Caudado/metabolismo , Centrifugação , Carvão Vegetal , Cocaína/farmacologia , Cães , Proteínas da Membrana Plasmática de Transporte de Dopamina , Técnicas In Vitro , Indicadores e Reagentes , Cinética , Ligantes , Proteínas do Tecido Nervoso/análise , Ligação Proteica , EstereoisomerismoRESUMO
Previously we have shown that the [125I]DEEP-labeled dopamine transporter from the rat nucleus accumbens has a higher apparent molecular weight than that from striatum. The present study confirms and extends these observations. Experiments with nucleus accumbens showed [125I]-DEEP to specifically bind to a protein with an apparent molecular weight of 76 kDa and with the pharmacological properties of the dopamine transporter. In exoglycosidase studies, treatment with neuraminidase, but not alpha-mannosidase, reduced the apparent molecular weight of the dopamine transporter from both the striatum and nucleus accumbens; however, a difference in the apparent molecular weight was still observed. N-Glycanase treatment, on the other hand, did reduce the apparent molecular weight of the dopamine transporters from the two regions to a similar value, approximately 56 kDa. In radioligand binding studies examining the effect of partial deglycosylation on striatal dopamine transporters, neuraminidase did not affect specific [3H]WIN 35,428 binding at 4 and 40 nM concentrations. In conclusion, the present study demonstrates that the difference in the apparent molecular weight of the dopamine transporter from these two regions is due to a difference in glycosylation and that the dopamine transporter from both regions contains similar amounts of sialic acid in their carbohydrate structure. Furthermore, the present data also indicate that the polypeptide portion of the dopamine transporter from both regions could be the same gene product.
Assuntos
Proteínas de Transporte/metabolismo , Corpo Estriado/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Núcleo Accumbens/metabolismo , Animais , Azidas , Cocaína/análogos & derivados , Proteínas da Membrana Plasmática de Transporte de Dopamina , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/farmacologia , Técnicas In Vitro , Masculino , Membranas/efeitos dos fármacos , Membranas/metabolismo , Neuraminidase/farmacologia , Piperazinas , Ratos , Ratos EndogâmicosRESUMO
The nature of methamphetamine-induced rapid and transient decreases in dopamine transporter activity was investigated. Regional specificity was demonstrated, since [3H]dopamine uptake was decreased in synaptosomes prepared from the striatum, but not nucleus accumbens, of methamphetamine-treated rats. Differences among effects on dopamine transporter activity and ligand binding were also observed, since a single methamphetamine administration decreased [3H]dopamine uptake without altering [3H]WIN35428 ([3H](-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane 1,5-naphthalenedisulfonate) binding in synaptosomes prepared 1 h after injection. Moreover, multiple methamphetamine injections caused a greater decrease in [3H]dopamine uptake than [3H]WIN35428 binding in synaptosomes prepared I h after dosing. Finally, decreases in [3H]dopamine uptake, but not [3H]WIN35428 binding, were partially reversed 24 h after multiple methamphetamine injections. Western blotting indicated that saline- and methamphetamine-affected dopamine transporters co-migrated on sodium dodecyl sulfate (SDS) gels at approximately 80 kDa, and that acute, methamphetamine-induced decreases in [3H]dopamine uptake were not due to loss of dopamine transporter protein. These findings demonstrate heretofore-uncharacterized features of the acute effect of methamphetamine on dopamine transporters.
Assuntos
Proteínas de Transporte/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Metanfetamina/farmacologia , Proteínas do Tecido Nervoso , Animais , Proteínas de Transporte/efeitos dos fármacos , Cocaína/análogos & derivados , Cocaína/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Masculino , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The dopamine transporter (DAT) in rat striatum was examined during postnatal development and aging after photolabeling with [125I]DEEP. The DAT-[125I]DEEP protein complex from adult rats (2 months) appeared as a broad diffuse band in SDS-PAGE gels with average apparent molecular mass of about 80,000 Da as previously found. However, the molecular mass was lower at birth (day 0) and at postnatal ages 4 and 14 days. In aged rats (104 weeks), the molecular mass was slightly higher than that found in young adults (60 days). In binding experiments with [3H]BTCP, there were age-related differences in Kd and Bmax with decreases in both Kd and Bmax found in aged rats. Treatment of photolabeled membranes with neuraminidase caused a reduction in DAT molecular mass, but age-related differences were maintained. Treatment with N-glycanase greatly reduced or eliminated the age-related differences. Several DAT peptide-specific polyclonal antibodies immunoprecipitated DAT-[125I]DEEP protein complex at different developmental ages. Taken together, these results suggest differential glycosylation of rat DAT occurs during postnatal development and aging; the increase is due to increases in the N-linked sugars rather than changes in either sialic acid content or the polypeptide.
Assuntos
Envelhecimento/metabolismo , Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Azidas/metabolismo , Proteínas de Transporte/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Dopamina/metabolismo , Agonistas de Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Feminino , Glicosilação , Cinética , Masculino , Dados de Sequência Molecular , Fenciclidina/análogos & derivados , Fenciclidina/metabolismo , Piperazinas/metabolismo , Gravidez , Estrutura Secundária de Proteína , Ratos , Ratos Sprague-Dawley , Ácidos Siálicos/análiseRESUMO
The dopamine transporter (DAT) has been implicated strongly in cocaine's reinforcing effects. Many derivatives of piperidine analogs of GBR 12909 have been developed and were found to be quite potent and selective for the DAT. In this regard, most of these derivatives were found to be much more selective for the DAT than conventional GBR compounds e.g. GBR 12909 when their selectivity was compared with the serotonin transporter (SERT). A brief structure-activity relationship (SAR) study has been carried out in the development of a novel photoaffinity ligand which illustrated the effect of the presence of a sterically bulky iodine atom next to the azido group in activity and selectivity for the DAT. This SAR study also led to the development of the compound 4 which is one of the most potent and selective blockers for the DAT known today. The photoaffinity ligand [125I]AD-96-129 was incorporated into the DAT molecule as was demonstrated by immunoprecipitation with serum 16 which is specific for DAT. This photolabeling was antagonized by DAT-specific blockers and was unaffected by specific SERT and norepinephrine transporter (NET) blockers indicating interaction of this novel ligand with the DAT.
Assuntos
Proteínas de Transporte/síntese química , Dopamina/síntese química , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Marcadores de Fotoafinidade/síntese química , Piperidinas/síntese química , Animais , Proteínas de Transporte/química , Proteínas de Transporte/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopamina/química , Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina , Inibidores da Captação de Dopamina/química , Desenho de Fármacos , Glicoproteínas de Membrana/farmacologia , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/farmacologia , Piperazinas/química , Piperidinas/química , Piperidinas/farmacologia , Ratos , Proteínas da Membrana Plasmática de Transporte de Serotonina , Relação Estrutura-AtividadeRESUMO
We investigated the functional relationship between the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein syntaxin 1A (syn 1A) and the dopamine transporter (DAT) by treating rat striatal tissue with Botulinum Neurotoxin C (BoNT/C) and co-transfecting syn 1A with DAT in non-neuronal cells, followed by analysis of DAT activity, phosphorylation, and regulation. Treatment of striatal slices with BoNT/C resulted in elevated dopamine (DA) transport Vmax and reduced DAT phosphorylation, while heterologous co-expression of syn 1A led to reduction in DAT surface expression and transport Vmax. Syn 1A was present in DAT immunoprecipitation complexes, supporting a direct or indirect interaction between the proteins. Phorbol ester regulation of DA transport activity was retained in BoNT/C-treated synaptosomes and syn 1A transfected cells, demonstrating that protein kinase C (PKC) and syn 1A effects occur through independent processes. These findings reveal a novel mechanism for regulation of DAT activity and phosphorylation, and suggest the potential for syn 1A to impact DA neurotransmission through effects on reuptake.
Assuntos
Toxinas Botulínicas/farmacologia , Corpo Estriado/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptor Cross-Talk/efeitos dos fármacos , Sintaxina 1/metabolismo , Animais , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Células LLC-PK1 , Masculino , Ésteres de Forbol/farmacologia , Ratos , Ratos Sprague-Dawley , Suínos , Sinaptossomos/metabolismo , Transfecção/métodosAssuntos
Proteínas de Transporte/metabolismo , Cocaína/farmacologia , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Fosfoproteínas/metabolismo , Animais , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Proteínas do Tecido Nervoso/metabolismo , Ésteres de Forbol/farmacologia , Fosfoproteínas/isolamento & purificação , Fosforilação , Proteínas Quinases/metabolismo , Ratos , Transtornos Relacionados ao Uso de Substâncias , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Binding domains on rat dopamine transporters for cocaine and 1-(2-diphenylmethoxy)ethyl-4-(3-phenylpropyl)piperazine compounds were identified using controlled proteolysis of photoaffinity-labeled protein and epitope-specific immunoprecipitation of the labeled fragments. Rat dopamine transporters were photoaffinity labeled with 1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido- 3-[125I]iodophenyl)ethyl]piperazine ([125I]DEEP) [a 1-(2-di- phenylmethoxy)ethyl-4-(3-phenylpropyl)piperazine analog] or 3 beta-(p-chlorophenyl)tropane-2 beta-carboxylic acid, 4'-azido-3'- [125I]iodophenylethyl ester ([125I]RTI 82) (a cocaine analog) and were gel purified to remove contaminating radioactivity. The resulting samples were treated with V8 protease or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptide maps generated with each enzyme were different for each of the ligands, suggesting that the ligands were incorporated into different regions of the protein. Identical peptide maps were generated from striatum- and nucleus accumbens-derived transporters, indicating that these polypeptides are highly similar in primary sequence. The proteolytic fragments generated by V8 protease were localized to specific domains of the protein using antipeptide antibodies corresponding to five different regions of the transporter. Fragments of 10 and 7 kDa from [125I]DEEP-labeled transporters were specifically immunoprecipitated with an antibody generated against amino acids 42-59 (near the first putative trans-membrane domain), whereas a 34-kDa fragment from [125I]RTI 82-labeled transporters was precipitated with three different sera corresponding to regions in the carboxyl-terminal two thirds of the protein. None of the V8 fragments smaller than 45 kDa, containing either photolabel, was altered in molecular mass by N-deglycosylation. The results indicate that photoincorporation of [125I]DEEP occurs in the amino half of the dopamine transporter, near the first two transmembrane helices, whereas [125I]RTI 82 labels the carboxyl-terminal region of the protein, between transmembrane domains 4 and 12.
Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Marcadores de Afinidade , Sequência de Aminoácidos , Animais , Azidas , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cocaína/análogos & derivados , Cocaína/metabolismo , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Endopeptidases/metabolismo , Imunoquímica , Técnicas In Vitro , Ligantes , Dados de Sequência Molecular , Núcleo Accumbens/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Fotoquímica , Piperazinas , RatosRESUMO
During the developmental cycle of Dictyostelium discoideum cyclic AMP functions as both a chemotactic signal for aggregation and a regulatory molecule during later events of differentiation. Morphological and biochemical data suggest that cAMP may direct cells during morphogenesis and differentiation. We utilized microtechniques to determine the stage- and cell-specific levels of the cAMP-dependent protein kinase, the probable intracellular cAMP receptor. Kinase activity was low and non-cAMP-dependent in amoebae and early aggregates but increased and became cAMP-dependent in aggregates after the formation of tight cell contacts. Maximum kinase activity and cAMP dependency occurred during the slug and culmination stages. The only differential distribution of the kinase within a single stage occurred during culmination when the activity in the stalks was approximately one-fourth of that in the prespore mass. Preliminary evidence indicates that this difference is not due to an inhibitor. In all other stages tested cAMP-dependent protein kinase activity was equal in prespore and prestalk cells.
Assuntos
AMP Cíclico/farmacologia , Dictyostelium/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Dictyostelium/enzimologia , Morfogênese , Oligopeptídeos/metabolismo , Solubilidade , Fatores de TempoRESUMO
The cell surface cAMP receptor of Dictyostelium discoideum exists as a doublet of low (D) and high (R) electrophoretic mobility forms, both of which are phosphorylated in vivo. The R form is phosphorylated in a ligand-independent manner, while conversion of the R to D forms, induced by the chemoattractant, is accompanied by at least a 4-fold increase in the level of phosphorylation. When cells are stimulated with saturating levels of cAMP, increased phosphorylation is detectable within 5 s and reaches maximum levels by 5 min with a t1/2 of 45 s. Dephosphorylation of receptor, initiated by removal of the stimulus, is detectable within 30 s, has a half-time of 2 min, and reaches a plateau by 20 min. At half-maximal occupancy, phosphorylation occurred more slowly than at saturation, t1/2 = 1.5 min, and remained at intermediate levels until the cAMP concentration was increased. Accompanying electrophoretic mobility shifts occurred in all cases with similar, though not identical, kinetics. Both phosphorylation and mobility shift were half-maximal at 5 nM cAMP and saturated at 100 nM. Estimation of the specific activity of each receptor form indicates that not all sites are phosphorylated during the R to D transition; at least half of the sites are phosphorylated after the transition is completed. The rate of incorporation of phosphates into the receptor, held in the D form by cAMP, was less than one-third the rate of ligand-induced incorporation starting with the R form and was approximately twice the basal rate of incorporation. These results are compatible with ligand-induced receptor phosphorylation being an early event in the adaptation of other cAMP-induced responses.
Assuntos
AMP Cíclico/metabolismo , Dictyostelium/metabolismo , Receptores de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Ligantes , FosforilaçãoRESUMO
Dopamine transporters (DATs) are members of the Na+- and Cl--dependent neurotransmitter and amino acid transporter family predicted by hydrophobicity analysis to have 12 transmembrane-spanning helices. The structure of DAT was studied using the photoaffinity compounds [125I]1-[2-(diphenylmethoxy)-ethyl]-4-[2-(4-azido-3-iodophenyl) ethyl] piperazine ([125I]DEEP), a 1-(2-diphenylmethoxy)-ethyl-4-(3-phenyl propyl)piperazine (GBR analog), and [125I]-3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82), a cocaine analog, which had been shown in a previous study to become incorporated into different regions of the DAT primary sequence. The proximity of the photolabeled binding sites to integral membrane structures was investigated by subjecting photolabeled membrane suspensions to limited proteolysis with trypsin and separately analyzing the resulting membranes and supernatants for the presence of photolabeled DAT fragments. Trypsin treatment of [125I] DEEP-labeled membranes generated labeled 45- and 14-kDa DAT fragments that immunoprecipitated with an epitope-specific antiserum generated against amino acids 42-59 near the first putative transmembrane domain, whereas [125I]RTI 82 was found in 32- and 16-kDa tryptic fragments that precipitated with an antiserum directed against a sequence near transmembrane domain 4 (amino acids 225-238). All of the photolabeled fragments were recovered in the protease-treated membranes, indicating that they possess integral membrane structures that prevent their release from the membrane as soluble forms. The size of the two smallest fragments in conjunction with their retention in the membrane suggests that incorporation of the photoaffinity ligands occurs in or near membrane spanning regions and delineates the maximum possible distance between the transmembrane structures, incorporated photolabel, and antibody epitopes. Carbohydrate analysis of the fragments identified sialic acids and N-linked oligosaccharides exclusively on the 45-kDa [125I]DEEP-labeled fragment, which, based on size, would be expected to contain four consensus glycosylation sites between putative transmembrane domains 3 and 4. Photoaffinity labeling after trypsin treatment of membranes showed that the larger but not the smaller fragments retain binding capacity, as the 45- and 32-kDa fragments were capable of becoming photolabeled. Binding of photoaffinity ligands at these fragments was displaced with the same pharmacology as that of intact DATs. These results verify numerous aspects of DAT structure and topology heretofore only predicted from theoretical considerations and extend our knowledge of DAT structure-function properties.
Assuntos
Proteínas de Transporte/metabolismo , Dopamina/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Marcadores de Afinidade , Animais , Azidas , Carboidratos/análise , Proteínas de Transporte/química , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Glicosilação , Ligantes , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Piperazinas , Ligação Proteica , Ratos , Tripsina/metabolismoRESUMO
Electron-spin-resonance measurements have been made on single crystals of uracil-beta-D-arabinofuranoside, which were irradiated by 4-0 MeV electrons at 77 K. At low temperatures, two radicals have been identified, one attributed to a hydrogen abstraction from 05' in the sugar moiety and the other to a radical anion located on the pyrimidine ring. The former is very unstable and seems to act as a precursor to other unidentified radical species stable at 77K. At room temperature, the main resonance is due to hydrogen addition to C5 and is probably produced by protonation of the anion. This same radical is also produced by X-irradiation at room temperature.