RESUMO
Interleukin-31 (IL-31) is involved in excessive development of cutaneous sensory nerves in atopic dermatitis (AD), leading to severe pruritus. We previously reported that PQA-18, a prenylated quinolinecarboxylic acid (PQA) derivative, is an immunosuppressant with inhibition of p21-activated kinase 2 (PAK2) and improves skin lesions in Nc/Nga mice as an AD model. In the present study, we investigate the effect of PQA-18 on sensory nerves in lesional skin. PQA-18 alleviates cutaneous nerve fiber density in the skin of Nc/Nga mice. PQA-18 also inhibits IL-31-induced sensory nerve fiber outgrowth in dorsal root ganglion cultures. Signaling analysis reveals that PQA-18 suppresses phosphorylation of PAK2, Janus kinase 2, and signal transducer and activator of transcription 3 (STAT3), activated by IL-31 receptor (IL-31R), resulting in inhibition of neurite outgrowth in Neuro2A cells. Gene silencing analysis for PAK2 confirms the requirement for STAT3 phosphorylation and neurite outgrowth elicited by IL-31R activation. LC/MS/MS analysis reveals that PQA-18 prevents the formation of PAK2 activation complexes induced by IL-31R activation. These results suggest that PQA-18 inhibits the IL-31 pathway through suppressing PAK2 activity, which suppresses sensory nerve outgrowth. PQA-18 may be a valuable lead for the development of a novel drug for pruritus of AD.
Assuntos
Ácidos Carboxílicos/farmacologia , Dermatite Atópica/tratamento farmacológico , Interleucinas/antagonistas & inibidores , Quinolinas/farmacologia , Células Receptoras Sensoriais/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Dermatite Atópica/metabolismo , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Interleucinas/metabolismo , Janus Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/patologia , Fatores de Crescimento Neural/metabolismo , Sistema Nervoso Periférico/efeitos dos fármacos , Sistema Nervoso Periférico/metabolismo , Prenilação de Proteína , Fator de Transcrição STAT3/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/metabolismo , Quinases Ativadas por p21/metabolismoRESUMO
Astrocytes release exosomes that regulate neuronal cell function. 1-methyl-4-phenylpyridinium (MPP+) is a well-known neurotoxin used to induce cell death in in vitro Parkinson's disease models, and microRNA (miRNA) transferred by released exosomes can regulate its mechanisms. Here, we demonstrated that exosomes released from normal astrocytes (ADEXs), but not exosomes derived from MPP+-stimulated astrocytes (MPP+-ADEXs), significantly attenuate MPP+-induced cell death in SH-SY5Y cells and primary mesencephalic dopaminergic neuron cultures, and reduce expression of mitogen-activated protein kinase kinase 4 (MKK4), an important upstream kinase in the c-Jun N-terminal kinase cell death pathway. Similar neuroprotective results were obtained from primary hippocampal neuron cultures, an in vitro glutamate excitotoxicity model. Through small-RNA sequencing of exosomal miRNA, we identified miR-200a-3p as the most down-regulated miRNA expressed in MPP+-ADEXs. miRNA target analysis and reporter assay confirmed that miR-200a-3p targets MKK4 through binding to two independent sites on the 3'-UTR of Map2k4/MKK4 mRNA. Treatment with miR-200a-3p mimic suppressed both MKK4 mRNA and protein expressions, and attenuated cell death in MPP+-treated SH-SY5Y cells and glutamate-treated hippocampal neuron cultures. Our results suggest that normal astrocytes release miR-200a-3p which exhibits a neuroprotective effect through down-regulation of MKK4.
Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Apoptose/fisiologia , Astrócitos/efeitos dos fármacos , Exossomos/metabolismo , MAP Quinase Quinase 4/metabolismo , MicroRNAs/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Astrócitos/metabolismo , Materiais Biomiméticos/farmacologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Exossomos/efeitos dos fármacos , Células HEK293 , Humanos , MAP Quinase Quinase 4/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The development of neuroprotective agents is necessary for the treatment of neurodegenerative diseases. Here, we report PQA-11, a prenylated quinolinecarboxylic acid (PQA) derivative, as a potent neuroprotectant. PQA-11 inhibits glutamate-induced cell death and caspase-3 activation in hippocampal cultures, as well as inhibits N-Methyl-4-phenylpyridinium iodide- and amyloid ß1-42-induced cell death in SH-SY5Y cells. PQA-11 also suppresses mitogen-activated protein kinase kinase 4 (MKK4) and c-jun N-terminal kinase (JNK) signaling activated by these neurotoxins. Quartz crystal microbalance analysis and in vitro kinase assay reveal that PQA-11 interacts with MKK4, and inhibits its sphingosine-induced activation. The administration of PQA-11 by intraperitoneal injection alleviates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced degeneration of nigrostriatal dopaminergic neurons in mice. These results suggest that PQA-11 is a unique MKK4 inhibitor with potent neuroprotective effects in vitro and in vivo. PQA-11 may be a valuable lead for the development of novel neuroprotectants.
Assuntos
Ácidos Carboxílicos/farmacologia , MAP Quinase Quinase 4/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Prenilação/efeitos dos fármacos , Quinolinas/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Tumoral , Humanos , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Prenilação/fisiologiaRESUMO
Reactive oxygen species (ROS) are implicated in the modulation of diverse processes including immune responses. To evaluate the effects of metabolic ROS produced by mitochondria on B-cell function and development, we created transgenic (Tg) mice expressing a phosphorylation-defective mutant of succinate dehydrogenase A in B cells (bSDHAY215F ). Splenic B cells in male, but not female, bSDHAY215F mice produced three times more ROS than those in the control mice, and had decreased production of IgM, IgG1 , and IgG3 , and affinity maturation of IgG1 against T-cell-dependent antigens. Following immunization, the male bSDHAY215F mice further displayed suppressed germinal center (GC) formation, and proliferation of GC B cells. Signaling analysis revealed defects in the intrinsic BCR responses, such as activation of Lyn, Btk, and PLCγ2, thus resulting in reduced intracellular Ca2+ mobilization. Notably, the expression levels of B-cell co-receptor CD19 and its interaction with Lyn after BCR ligation were significantly reduced in B cells from male bSDHAY215F mice. These results suggest that mitochondrial ROS suppress humoral immune responses through reduction of CD19 expression and resultant BCR signaling in B cells. Therefore, B-cell immunity may be more labile to oxidative stress in male mice than in female mice.
Assuntos
Antígenos CD19/metabolismo , Linfócitos B/imunologia , Complexo II de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Antígenos CD19/genética , Células Cultivadas , Complexo II de Transporte de Elétrons/genética , Feminino , Imunidade Humoral , Terapia de Imunossupressão , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Fosforilação/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/genéticaRESUMO
Development of new immunosuppressing agents is necessary in organ transplantation or immune diseases. Because Ppc-1 exhibits a suppressing effect on interleukin-2 (IL2) production in Jurkat cells, we synthesized and screened Ppc-1 derivatives that preserve prenylated quinolinecarboxylic acid (PQA) structure, and identified compound 18 (PQA-18) as a novel molecule with immunosuppressing effect. PQA-18 suppressed not only IL2 but also IL4, IL6, and tumor necrosis factor-α production in human peripheral lymphocytes without affecting cell viability. Two-dimensional gel electrophoresis analysis and in vitro kinase assay revealed that PQA-18 inhibits kinase activity of p21-activated kinase 2 (PAK2). Administration of PQA-18 by intraperitoneal injection suppressed the population of a subset of regulatory T cells and the immunoglobulin (Ig) production against T cell-dependent antigens in mice. Treatment with the PQA-18 ointment on Nc/Nga mice, a model of human atopic dermatitis, improved skin lesions and serum IgE levels. These results suggest that PQA-18 is a unique PAK2 inhibitor with potent immunosuppressing effects in vitro and in vivo. PQA-18 may be a valuable lead for the development of novel immunosuppressants.
Assuntos
Ácidos Carboxílicos/farmacologia , Imunidade Celular/fisiologia , Imunossupressores/farmacologia , Prenilação/fisiologia , Quinolinas/farmacologia , Quinases Ativadas por p21/metabolismo , Animais , Ácidos Carboxílicos/química , Dermatite/tratamento farmacológico , Dermatite/imunologia , Dermatite/metabolismo , Relação Dose-Resposta a Droga , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/imunologia , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prenilação/efeitos dos fármacos , Prenilação/imunologia , Quinolinas/química , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Ácidos Tri-Iodobenzoicos/farmacologia , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/imunologiaRESUMO
We have shown marked promotion of both cluster growth and neuronal specification in pluripotent P19 cells with overexpression of solute carrier 38a1 (Slc38a1), which is responsible for membrane transport of glutamine. In this study, we evaluated pharmacological profiles of the green tea amino acid ingredient theanine, which is a good substrate for glutamine transporters, on proliferation and neuronal specification in neural progenitor cells from embryonic rat neocortex. Sustained exposure to theanine, but not glutamine, accelerated the growth of neurospheres composed of proliferating cells and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reducing activity at concentrations of 1-100 µM in undifferentiated progenitor cells. Such prior exposure to theanine promoted spontaneous and induced commitment to a neuronal lineage with concomitant deteriorated astroglial specification. Selective upregulation was seen in the expression of Slc38a1 in progenitor cells cultured with theanine. Similarly significant increases in cluster growth and MTT reducing activity were found in P19 cells cultured with theanine for 4 days. Luciferase activity was doubled in a manner sensitive to the deletion of promoter regions in P19 cells with a luciferase reporter plasmid of the Slc38a1 promoter after sustained exposure to theanine for 4 days. Overexpression of X-box binding protein-1 led to a marked increase in luciferase activity in P19 cells transfected with the Slc38a1 reporter plasmid. These results suggest that theanine accelerates cellular proliferation and subsequent neuronal specification through a mechanism relevant to upregulation of Slc38a1 gene in undifferentiated neural progenitor cells.
Assuntos
Sistema A de Transporte de Aminoácidos/genética , Diferenciação Celular/genética , Glutamatos/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Regulação para Cima , Animais , Proliferação de Células/genética , Células Cultivadas , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , RNA Mensageiro/genética , Ratos , Ratos WistarRESUMO
INTRODUCTION: Early detection and/or prediction of metastasis provide more prognostic relevance than local recurrence. Direct spread into the peritoneum is frequently found in pancreatic cancer patients, but positron emission tomography (PET) with 2-deoxy-2-fluoro-d-glucose (FDG) is not useful for identifying such metastasis. We investigated a method to enhance FDG accumulation using AsPC-1 human ascites tumor cells. METHODS: (14)C-FDG accumulation was assessed under the following conditions: 1) characteristics of (14)C-FDG transport were examined using phloridzin, a Na(+)-free buffer, and various hexoses, and 2) accumulation of (14)C-FDG was measured in cells that were pretreated with hexose for various time periods, and activity of 6-phosphofructo-1-kinase (PFK-1) was assayed. RESULTS: (14)C-FDG transport into AsPC-1 cells was mediated primarily by a Na(+)-independent transport mechanism. Aldohexoses such as d-glucose, D-mannose, and D-galactose inhibited (14)C-FDG transport. Cells pretreated with d-glucose, D-mannose, or D-fructose exhibited augmented (14)C-FDG accumulation. Pretreatment with higher concentrations of D-glucose or D-fructose tended to increase PFK-1 activity. CONCLUSIONS: Very little information has been published about the association between PFK-1 and FDG accumulation, and we confirmed the impacts of various hexoses on the activity of PFK-1 and FDG accumulation in AsPC-1 cells. Clarifying the relevance of PFK-1 in FDG accumulation will contribute to developing new features of FDG-PET, because PFK-1 is the main regulator of glycolysis.
Assuntos
Detecção Precoce de Câncer/métodos , Fluordesoxiglucose F18/metabolismo , Glicólise , Neoplasias Pancreáticas/patologia , Ascite/patologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Glicólise/efeitos dos fármacos , Hexoses/farmacologia , Humanos , Metástase Neoplásica , Fosfofrutoquinase-1/metabolismo , Tomografia por Emissão de PósitronsRESUMO
Mitochondria play a key role in diverse processes including ATP synthesis and apoptosis. Mitochondrial function can be studied using inhibitors of respiration, and new agents are valuable for discovering novel mechanisms involved in mitochondrial regulation. Here, we screened small molecules derived from slime molds and other microorganisms for their effects on mitochondrial oxygen consumption. We identified Ppc-1 as a novel molecule which stimulates oxygen consumption without adverse effects on ATP production. The kinetic behavior of Ppc-1 suggests its function as a mitochondrial uncoupler. Serial administration of Ppc-1 into mice suppressed weight gain with no abnormal effects on liver or kidney tissues, and no evidence of tumor formation. Serum fatty acid levels were significantly elevated in mice treated with Ppc-1, while body fat content remained low. After a single administration, Ppc-1 distributes into various tissues of individual animals at low levels. Ppc-1 stimulates adipocytes in culture to release fatty acids, which might explain the elevated serum fatty acids in Ppc-1-treated mice. The results suggest that Ppc-1 is a unique mitochondrial regulator which will be a valuable tool for mitochondrial research as well as the development of new drugs to treat obesity.
Assuntos
Dictyosteliida/metabolismo , Mitocôndrias/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Redução de Peso/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Ácidos Graxos/metabolismo , Feminino , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacosRESUMO
Mitochondrial uncoupling is one of the therapeutic strategies used to control energy metabolism in various metabolic diseases and in obesity. Ppc-1 (1), a prenylated quinolinecarboxylic acid isolated from cellular slime molds, shows uncoupling activity in vitro and anti-obesity activity in vivo. In this study, we synthesized Ppc-1 (1) and its derivatives, and revealed the structure-activity relationship of uncoupling activities. The triprenylated compound 18 showed mitochondrial uncoupling activity that was more potent than that of Ppc-1 (1). Compound 18 also suppressed weight gain in mice without undesired effects such as lesions on tissues. These results indicate that compound 18 could be used as a seed compound for new anti-obesity drugs.
Assuntos
Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/farmacologia , Obesidade/tratamento farmacológico , Ácidos Quinolínicos/síntese química , Ácidos Quinolínicos/farmacologia , Animais , Fármacos Antiobesidade/química , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Obesidade/metabolismo , Prenilação , Ácidos Quinolínicos/química , Relação Estrutura-Atividade , Desacopladores/síntese química , Desacopladores/química , Desacopladores/farmacologiaRESUMO
We have shown that mitochondrial c-Src regulates reactive oxygen species (ROS) production by phosphorylating the succinate dehydrogenase A of respiratory complex II (CxII). To elucidate the molecular mechanisms underlying ROS production regulated by c-Src in the CxII, we investigated the CxII protein complex derived from cells treated with Src family kinase inhibitor PP2. We identified flotillin-1 as a c-Src target that prevents ROS production from CxII. Phosphorylation-site analysis suggests Tyr56 and Tyr149 on flotillin-1 as sites for phosphorylation by c-Src. A comparison of cells expressing flotillin-1 and its phosphorylation defective mutants confirms the requirement for flotillin-1 phosphorylation for its interaction with CxII and subsequent reduction in ROS production. Our findings suggest a critical role of flotillin-1 in ROS production mediated by c-Src.
Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Quinases da Família src/metabolismo , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Complexo II de Transporte de Elétrons/metabolismo , Humanos , Proteínas de Membrana/química , Mitocôndrias/enzimologia , Fosforilação , Tirosina/metabolismoRESUMO
BPA used in BNCT has a similar structure to some essential amino acids and is transported into tumor cells by amino acid transport systems. Previous study groups have tried various techniques of loading BPA to increase intracellular boron concentration. CHO-K1 cells demonstrate system L (LAT1) activity and are suitable for specifying the transport system of a neutral amino acid. In this study, we examined the intracellular accumulation of boron in CHO-K1 cells by amino acid transport control, which involves co-loading with L-type amino acid esters. Intracellular boron accumulation in CHO-K1 cells showed the greatest increased upon co-loading 1.0mM BPA, with 1.0mM l-Tyr-O-Et and incubating for 60min. This increase is caused by activation of a system L amino acid exchanger between BPA and l-Tyr. The amino acid esters are metabolized to amino acids by intracellular hydrolytic enzymes that increase the concentrations of intracellular amino acids and stimulate exchange transportation. We expect that this amino acid transport control will be useful for enhancing intracellular boron accumulation.
Assuntos
Compostos de Boro/farmacocinética , Terapia por Captura de Nêutron de Boro/métodos , Portadores de Fármacos/farmacocinética , Fenilalanina/análogos & derivados , Tirosina/farmacocinética , Sistemas de Transporte de Aminoácidos/farmacocinética , Animais , Compostos de Boro/administração & dosagem , Compostos de Boro/química , Células CHO , Cricetinae , Cricetulus , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Taxa de Depuração Metabólica , Fenilalanina/administração & dosagem , Fenilalanina/química , Fenilalanina/farmacocinética , Tirosina/administração & dosagem , Tirosina/químicaRESUMO
BACKGROUND: We have previously shown marked upregulation of the mRNA and corresponding protein for the cellular motor molecule myosin VI (Myo6) after an extremely traumatic stress experience, along with a delayed decrease in 5-bromo-2'-deoxyuridine incorporation in the murine hippocampus, a brain structure believed to undergo adult neurogenesis. In this study, we investigated the role of Myo6 in both proliferation and differentiation in pluripotent P19 cells by using stable transfection and RNA interference techniques. METHODOLOGY/PRINCIPAL FINDINGS: Stable overexpression of Myo6 not only led to significant inhibition of the reducing activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the size of clustered aggregates in P19 cells, but also resulted in selectively decreased mRNA expression of the repressor type proneural gene Hes5 without affecting the expression of neuronal and astroglial marker proteins. In P19 cells transfected with Myo6 siRNA, by contrast, a significant increase was found in the size of aggregate and MTT reduction along with increased Sox2 protein levels, in addition to marked depletion of the endogenous Myo6 protein. In C6 glioma cells, however, introduction of Myo6 siRNA induced a drastic decrease in endogenous Myo6 protein levels without significantly affecting MTT reduction. The Ca(2+) ionophore A23187 drastically increased the luciferase activity in P19 cells transfected with a Myo6 promoter reporter plasmid, but not in HEK293, Neuro2A and C6 glioma cells transfected with the same reporter. CONCLUSIONS/SIGNIFICANCE: These results suggest that Myo6 may play a predominant pivotal role in the mechanism underlying proliferation without affecting differentiation to progeny lineages in pluripotent P19 cells.
Assuntos
Diferenciação Celular/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Células-Tronco Pluripotentes/metabolismo , Análise de Variância , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Primers do DNA/genética , Luciferases , Camundongos , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Repressoras/metabolismo , Sais de Tetrazólio , Tiazóis , TransfecçãoRESUMO
BACKGROUND: We previously demonstrated the functional expression in newborn rat neocortical astrocytes of glutamine transporter (GlnT = slc38a1) believed to predominate in neurons over astroglia in the brain. In order to evaluate the possible role of this transporter in neurogenesis, we attempted to establish stable transfectants of GlnT in mouse embryonal carcinoma P19 cells endowed to proliferate for self-renewal and differentiate into progeny cells such as neurons and astroglia, in addition to in vitro pharmacological profiling of the green tea ingredient theanine, which is shown to be a potent inhibitor of glutamine transport mediated by GlnT in cultured neurons and astroglia. METHODOLOGY/PRINCIPAL FINDINGS: The full-length coding region of rat GlnT was inserted into a vector for gene transfection along with selection by G418, followed by culture with all-trans retinoic acid under floating conditions and subsequent dispersion for spontaneous differentiation under adherent conditions. Stable overexpression of GlnT led to marked increases in the size of round spheres formed during the culture for 4 days and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction, with concomitant promotion of subsequent differentiation into cells immunoreactive for a neuronal marker protein. In these stable GlnT transfectants before differentiation, drastic upregulation was seen for mRNA expression of several proneural genes with a basic helix-loop-helix domain such as NeuroD1. Although a drastic increase was seen in NeuroD1 promoter activity in stable GlnT transfectants, theanine doubled NeuroD1 promoter activity in stable transfectants of empty vector (EV), without affecting the promoter activity already elevated in GlnT transfectants. Similarly, theanine promoted cellular proliferation and neuronal differentiation in stable EV transfectants, but failed to further stimulate the acceleration of both proliferation and neuronal differentiation found in stable GlnT transfectants. CONCLUSIONS/SIGNIFICANCE: GlnT would promote both proliferation and neuronal differentiation through a mechanism relevant to the upregulation of particular proneural genes in undifferentiated P19 cells.
Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco de Carcinoma Embrionário/citologia , Células-Tronco de Carcinoma Embrionário/metabolismo , Neurogênese/fisiologia , Sistema A de Transporte de Aminoácidos/genética , Animais , Northern Blotting , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Cromatografia Líquida de Alta Pressão , Imuno-Histoquímica , Camundongos , Neurogênese/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr(193) of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr(215) of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.
Assuntos
Transporte de Elétrons/fisiologia , Mitocôndrias/metabolismo , Proteínas Tirosina Quinases/metabolismo , Quinases da Família src/metabolismo , Animais , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Sobrevivência Celular , Complexo II de Transporte de Elétrons/metabolismo , Humanos , Camundongos , Mitocôndrias/enzimologia , NADH Desidrogenase/metabolismo , Consumo de Oxigênio , FosforilaçãoRESUMO
The underlying mechanisms are still unclear for the neuroprotective properties of nicotine to date, whereas we have shown functional expression of nicotinic acetylcholine receptors (nAChRs) responsible for the influx of extracellular Ca(2+) in cultured rat cortical astrocytes. In this study, we investigated the possible involvement of astrocytic nAChRs in the neuroprotection by this agonist. Exposure to nicotine predominantly induced mRNA expression of glial cell line-derived neurotrophic factor (GDNF) among the different neurotrophic factors examined in cultured astrocytes, in a manner sensitive to nAChR antagonists, nifedipine, and aCa(2+) chelator. Nicotine significantly increased GDNF in a concentration-dependent manner in cultured astrocytes but not in neurons or neural progenitors even at the highest concentration used. In cultured astrocytes, a transient increase was seen in the expression of mRNA and corresponding protein for GDNF during sustained exposure to nicotine for 24 hr. Cytotoxicity mediated by oxidative, calcium, mitochondrial, or endoplasmic reticulum stress was invariably protected against in cortical neurons cultured with conditioned medium from astrocytes previously exposed to nicotine, and preincubation with the anti-GDNF antibody reduced the neuroprotection by conditioned medium from astrocytes exposed to nicotine. Intraperitoneal administration of nicotine transiently increased the number of cells immunoreactive for both GDNF and glial fibrillary acidic protein in rat cerebral cortex. These results suggest that astrocytic nAChRs play a role in the neuroprotection against different cytotoxins after predominant upregulation of GDNF expression through a mechanism relevant to the acceleration of extracellular Ca(2+) influx in rat brain in a particular situation.
Assuntos
Astrócitos/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Nicotina/metabolismo , Nicotina/farmacologia , Agonistas Nicotínicos/metabolismo , Agonistas Nicotínicos/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
We have previously demonstrated the functional expression of glutamine (Gln) transporter (GlnT) believed to predominate in neurons for the neurotransmitter glutamate pool by rat neocortical astrocytes devoid of neuronal marker expression, with exacerbated vulnerability to oxidative stress after transient overexpression. To evaluate molecular mechanisms underlying the exacerbation, we established stable GlnT transfectants in rat astrocytic C6 glioma cells. In two different clones of stable transfectants with increased intracellular Gln levels, exposure to hydrogen peroxide (H(2)O(2)) and A23187, but not to tunicamycin or 2,4-dinitrophenol, led to significant exacerbation of the cytotoxicity compared to cells with empty vector (EV). Stable GlnT overexpression led to a significant increase in heme oxygenase-1 protein levels in a manner sensitive to H(2)O(2), whereas H(2)O(2) was significantly more effective in increasing NO(2) accumulation and reactive oxygen species (ROS) generation in stable GlnT transfectants than in EV cells. Moreover, exposure to A23187 led to a more effective increase in the generation of ROS in stable GlnT transfectants than in stable EV transfectants. These results suggest that GlnT may play a role in the mechanisms underlying the determination of cellular viability in astrocytes through modulation of intracellular ROS generation.
Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Estresse Oxidativo , Sistema A de Transporte de Aminoácidos/genética , Animais , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Peróxido de Hidrogênio/farmacologia , Óxido Nítrico/biossíntese , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
INTRODUCTION: High expression of the system L amino acid transporter has been observed in clinically important tissues including tumors and the blood-brain barrier. We examined amino acid transport system L selectivity of (14)C(U)-L-tyrosine ((14)C-Tyr), (125)I-4-iodo-L-meta-tyrosine (4-(125)I-mTyr), (125)I-6-iodo-L-meta-tyrosine (6-(125)I-mTyr), (125)I-3-iodo-α-methyl-L-tyrosine ((125)I-IMT) and (125)I-3-iodo-L-tyrosine (3-(125)I-Tyr) using Chinese hamster ovary cells (CHO-K1). METHODS: Cells in the exponential growth phase were incubated with 18.5 kBq of labeled amino acid in 2 mL of phosphate-buffered saline-based uptake solution and an uptake solution with/without Na(+) at 37°C or 4°C. We examined the effects of the following compounds (1.0 mM) on transport: 2-(methylamino)isobutyric acid (a specific inhibitor of system A, in Na(+)-containing uptake solution); 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L, in Na(+)-free uptake solution); sodium azide and 2,4-dinitrophenol (NaN(3) and DNP, inhibitors of the generation of adenosine triphosphate); p-aminohippurate and tetraethylammonium (PAH and TEA, inhibitors of organic anion and cation transporters); and L- and D-isomers of natural amino acids. RESULTS: (14)C-Tyr exhibited affinity for systems L, A and ASC. 4-(125)I-mTyr and 3-(125)I-Tyr exhibited high specificity for system L, whereas 6-(125)I-mTyr and (125)I-IMT exhibited affinity for both systems L and ASC. Uptake of 4-(125)I-mTyr was markedly reduced by incubation at 4 °C, and was not significantly inhibited by NaN(3), DNP, PAH or TEA. The inhibition profiles of the L- and D-isomers of natural amino acids indicated that system L mediates the transport of 4-(125)I-mTyr. CONCLUSIONS: 4-(125)I-mTyr exhibited the greatest system L specificity (93.46 ± 0.13%) of all of the tested amino acids.
Assuntos
Monoiodotirosina/química , Monoiodotirosina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Células CHO , Proliferação de Células , Cricetinae , Cricetulus , Radioisótopos do Iodo , Cinética , Estereoisomerismo , Especificidade por SubstratoRESUMO
INTRODUCTION: Transport of the amino acid analog (123)I-3-iodo-alpha-methyl-L-tyrosine, which is used in clinical SPECT imaging, occurs mainly via L-type amino acid transporter type 1 (LAT1; an amino acid exchanger). As LAT1 is highly expressed in actively proliferating tumors, we made a preliminary investigation of the effects of amino acid esters on enhancement of (125)I-3-iodo-alpha-methyl-L-tyrosine (IMT) uptake via LAT1 in Chinese hamster ovary (CHO-K1) cells. METHODS: Because the sequence of the CHO-K1 LAT1 gene is not available, we confirmed LAT1 expression through IMT (18.5 kBq) uptake mechanisms using specific inhibitors. L-Gly, L-Ser, L-Leu, L-Phe, L-Met, L-Tyr, D-Tyr, L-Val and L-Lys ethyl/methyl esters were tested in combination with IMT. Time-course studies over a 3-h period were conducted, and the concentration dependence of L-Tyr ethyl and methyl esters (0.001 to 10 mM) in combination with IMT was also examined. For a proof of de-esterification of L- and D-Tyr ethyl and methyl esters in the cells (by enzymatic attack or other cause), the concentration of L- and D-Tyr was analyzed by high-performance liquid chromatography of the esters in phosphate buffer (pH 7.4) and cell homogenates at 37 degrees C or under ice-cold conditions. RESULTS: Inhibition tests suggested that LAT1 is involved in IMT uptake by CHO-K1 cells. Co-administration of 1 mM of l-Tyr ethyl or methyl ester with IMT produced the greatest enhancement. The de-esterification reaction was stereo selective and temperature dependent in the homogenate. De-esterification kinetics were very fast in the homogenate and very slow in the phosphate buffer. CONCLUSIONS: The L-Tyr ethyl or methyl esters were the most effective enhancers of IMT uptake into CHO-K1 cells and acted by trans-stimulation of the amino acid exchange function of LAT1. This result suggests that de-esterification in the cells may be caused by enzymatic attack. We will use IMT and L-Tyr ethyl or methyl esters to examine LAT1 function in tumor cells or tissues in vivo.
Assuntos
Ésteres/química , Ésteres/farmacologia , Metiltirosinas/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Células CHO , Extratos Celulares , Cricetinae , Cricetulus , Esterificação , Ésteres/metabolismo , Hidrólise , Transportador 1 de Aminoácidos Neutros Grandes/metabolismoRESUMO
INTRODUCTION: We examined 3-[(123)I]iodo-alpha-methyl-L-tyrosine ([(123)I]IMT) uptake and inhibition by amino acids and amino acid-like drugs in the human DLD-1 colon cancer cell line, to discuss correlation between the inhibition effect and structure. METHODS: Expression of relevant neutral amino acid transporters was examined by real-time PCR with DLD-1 cells. The time course of [(125)I]IMT uptake, contributions of transport systems, concentration dependence and inhibition effects by amino acids and amino acid-like drugs (1 mM) on [(125)I]IMT uptake were examined. RESULTS: Expression of system L (4F2hc, LAT1 and LAT2), system A (ATA1, ATA2) and system ASC (ASCT1) was strongly detected; system L (LAT3, LAT4) and MCT8 were weakly detected; and B(0)AT was not detected. [(125)I]IMT uptake in DLD-1 cells involved Na(+)-independent system L primarily and Na(+)-dependent system(s). Uptake of [(125)I]IMT in Na(+)-free buffer followed Michaelis-Menten kinetics, with a K(m) of 78 microM and V(max) of 333 pmol/10(6) cells per minute. Neutral D- and L-amino acids with branched or aromatic large side chains inhibited [(125)I]IMT uptake. Tyrosine analogues, tryptophan analogues, L-phenylalanine and p-halogeno-L-phenylalanines, and gamma amino acids [including 3,4-dihydroxy-L-phenylalanine (L-DOPA), DL-threo-beta-(3,4-dihydroxyphenyl)serine (DOPS), 4-[bis(2-chloroethyl)amino]-L-phenylalanine and 1-(aminomethyl)-cyclohexaneacetic acid] strongly inhibited [(125)I]IMT uptake, but L-tyrosine methyl ester and R(+)/S(-)-baclofen weakly inhibited uptake. The substrates of system ASC and A did not inhibit [(125)I]IMT uptake except L-serine and D/L-cysteine. CONCLUSIONS: [(125)I]IMT uptake in DLD-1 cells involves mostly LAT1 and its substrates' (including amino acid-like drugs derived from tyrosine, tryptophan and phenylalanine) affinity to transport via LAT1. Whether transport of gamma amino acid analogues is involved in LAT1 depends on the structure of the group corresponding to the amino acid residue. Beta-hydroxylation may confer reduction of transport affinity of tyrosine analogues via LAT1.
Assuntos
Aminoácidos/química , Aminoácidos/farmacologia , Neoplasias do Colo/patologia , Metiltirosinas/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Relação Estrutura-AtividadeRESUMO
OBJECTIVES: Almost all D-glucose (GLU) filtered through the glomerulus is reabsorbed by the renal proximal tubules, whereas a high portion of 2-[18F]fluoro-2-deoxy-D-glucose [(18F)FDG] is excreted through the urine. However, [18F]FDG is not entirely excreted in the urine suggesting that it may be partially reabsorbed by the proximal tubules. The purpose of this study was to compare the time course of transcellular transport of administered [14C] labeled FDG ([14C]FDG) with that of [14C] labeled GLU ([14C]GLU) using the kidney epithelial cell line, LLC-PK1. METHODS: Transcellular transport of [14C]FDG and [14C]GLU by LLC-PK1 cells was measured in Na+-containing or Na+-free Dulbecco's phosphate-buffered saline [PBS(+) and PBS(-), respectively] in the presence or absence of phlorizin, phloretin, probenecid, or tetraethylammonium bromide inhibitors that predominantly inhibit sodium-dependent glucose transporters (SGLTs), sodium-independent glucose transporters, organic anion transporters, and organic cation transporters, respectively. RESULTS: When assayed in PBS(+), less [14C]FDG than [14C]GLU was reabsorbed by the proximal tubular cells over the entire incubation time. Reabsorption of [14C]FDG was mediated mainly by SGLT at early time points in the incubation, whereas high reabsorption of [14C]GLU was mediated by both SGLT and glucose transporter over 90 min of incubation. Secretion of [14C]FDG also tended to be slightly higher than that of [14C]GLU over 90 min of incubation. CONCLUSION: Transcellular transport of [14C]FDG over time by LLC-PK1 cells was clarified. The polarized distribution of transcellular transporters of [14C]FDG and [14C]GLU in LLC-PK1 cells differs.