RESUMO
Cellular metabolism contributes to cell fate decisions. Bioenergetic profiling can therefore provide considerable insights into cellular identity and specification. Given the current importance of human pluripotent stem cells (hPSCs) for biomedical applications, assessing the bioenergetic properties of hPSCs and derivatives can unveil relevant mechanisms in the context of development biology and molecular disease modeling. Here, we describe a method to facilitate bioenergetic profiling of hPSCs in a reproducible and scalable manner. After simultaneous assessment of mitochondrial respiration and glycolytic capacity using Seahorse XFe96 Analyzer, we measure lactate concentration in the cellular media. Finally, we normalize the values based on DNA amount. We describe the procedures with specific requirements related to hPSCs . However, the same protocol can be easily adapted to other cell types, including differentiated progenies from hPSCs .
Assuntos
Mitocôndrias/metabolismo , Biologia Molecular/métodos , Células-Tronco Pluripotentes/metabolismo , Antimicina A/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Técnicas de Cultura de Células/métodos , DNA/análise , Metabolismo Energético/efeitos dos fármacos , Humanos , Ácido Láctico/análise , Mitocôndrias/efeitos dos fármacos , Oligomicinas/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Rotenona/farmacologiaRESUMO
Plant respiration can theoretically be fueled by and dependent upon an array of central metabolism components; however, which ones are responsible for the quantitative variation found in respiratory rates is unknown. Here, large-scale screens revealed 2-fold variation in nighttime leaf respiration rate (RN) among mature leaves from an Arabidopsis (Arabidopsis thaliana) natural accession collection grown under common favorable conditions. RN variation was mostly maintained in the absence of genetic variation, which emphasized the low heritability of RN and its plasticity toward relatively small environmental differences within the sampling regime. To pursue metabolic explanations for leaf RN variation, parallel metabolite level profiling and assays of total protein and starch were performed. Within an accession, RN correlated strongly with stored carbon substrates, including starch and dicarboxylic acids, as well as sucrose, major amino acids, shikimate, and salicylic acid. Among different accessions, metabolite-RN correlations were maintained with protein, sucrose, and major amino acids but not stored carbon substrates. A complementary screen of the effect of exogenous metabolites and effectors on leaf RN revealed that (1) RN is stimulated by the uncoupler FCCP and high levels of substrates, demonstrating that both adenylate turnover and substrate supply can limit leaf RN, and (2) inorganic nitrogen did not stimulate RN, consistent with limited nighttime nitrogen assimilation. Simultaneous measurements of RN and protein synthesis revealed that these processes were largely uncorrelated in mature leaves. These results indicate that differences in preceding daytime metabolic activities are the major source of variation in mature leaf RN under favorable controlled conditions.
Assuntos
Aminoácidos/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Metabolismo dos Carboidratos , Escuridão , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Arabidopsis/crescimento & desenvolvimento , Metabolismo dos Carboidratos/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Respiração Celular/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Ecótipo , Cromatografia Gasosa-Espectrometria de Massas , Metaboloma/efeitos dos fármacos , Modelos Biológicos , Consumo de Oxigênio/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Fatores de TempoRESUMO
Thermotherapy, as a method of treating cancer, has recently attracted considerable attention from basic and clinical investigators. A number of studies and clinical trials have shown that thermotherapy can be successfully used as a therapeutic approach for various cancers. However, the effects of temperature on cancer bioenergetics have not been studied in detail with a real time, microplate based, label-free detection approach. This study investigates how changes in temperature affect the bioenergetics characteristics (mitochondrial function and glycolysis) of three colorectal cancer (CRC) cell lines utilizing the Seahorse XF96 technology. Experiments were performed at 32°C, 37°C and 42°C using assay medium conditions and equipment settings adjusted to produce equal oxygen and pH levels ubiquitously at the beginning of all experiments. The results suggest that temperature significantly changes multiple components of glycolytic and mitochondrial function of all cell lines tested. Under hypothermia conditions (32°C), the extracellular acidification rates (ECAR) of CRC cells were significantly lower compared to the same basal ECAR levels measured at 37°C. Mitochondrial stress test for SW480 cells at 37°C vs 42°C demonstrated increased proton leak while all other OCR components remained unchanged (similar results were detected also for the patient-derived xenograft cells Pt.93). Interestingly, the FCCP dose response at 37°C vs 42°C show significant shifts in profiles, suggesting that single dose FCCP experiments might not be sufficient to characterize the mitochondrial metabolic potential when comparing groups, conditions or treatments. These findings provide valuable insights for the metabolic and bioenergetic changes of CRC cells under hypo- and hyperthermia conditions that could potentially lead to development of better targeted and personalized strategies for patients undergoing combined thermotherapy with chemotherapy.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Glicólise , Mitocôndrias/metabolismo , Temperatura , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Hipotermia Induzida , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Oxigênio/metabolismo , Fenótipo , Estresse Fisiológico/efeitos dos fármacosRESUMO
Mitochondrial dysfunction contributes to the development of muscle disorders, including muscle wasting, muscle atrophy and degeneration. Despite the knowledge that oxidative stress closely interacts with mitochondrial dysfunction, the detailed mechanisms remain obscure. In this study, tert-butylhydroperoxide (t-BHP) was used to induce oxidative stress on differentiated C2C12 myotubes. t-BHP induced significant mitochondrial dysfunction in a time-dependent manner, accompanied by decreased myosin heavy chain (MyHC) expression at both the mRNA and protein levels. Consistently, endogenous reactive oxygen species (ROS) overproduction triggered by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), a mitochondrial oxidative phosphorylation inhibitor, was accompanied by decreased membrane potential and decreased MyHC protein content. However, the free radical scavenger N-acetyl-L-cysteine (NAC) efficiently reduced the ROS level and restored MyHC content, suggesting a close association between ROS and MyHC expression. Meanwhile, we found that both t-BHP and FCCP promoted the cleavage of optic atrophy 1 (OPA1) from the long form into short form during the early stages. In addition, the ATPase family gene 3-like 2, a mitochondrial inner membrane protease, was also markedly increased. Moreover, OPA1 knockdown in myotubes was accompanied by decreased MyHC content, whereas NAC failed to prevent FCCP-induced MyHC decrease with OPA1 knockdown, suggesting that ROS might affect MyHC content by modulating OPA1 cleavage. In addition, hydroxytyrosol acetate (HT-AC), an important compound in virgin olive oil, could significantly prevent t-BHP-induced mitochondrial membrane potential and cell viability loss in myotubes. Specifically, HT-AC inhibited t-BHP-induced OPA1 cleavage and mitochondrial morphology changes, accompanied by improvement on mitochondrial oxygen consumption capacity, ATP productive potential and activities of mitochondrial complex I, II and V. Moreover, both t-BHP- and FCCP-induced MyHC decrease was sufficiently inhibited by HT-AC. Taken together, our data provide evidence indicating that mitochondrial dysfunction-associated OPA1 cleavage may contribute to muscle degeneration, and olive oil compounds could be effective nutrients for preventing the development of muscle disorders.
Assuntos
Acetatos/farmacologia , Catecóis/farmacologia , GTP Fosfo-Hidrolases/genética , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Cadeias Pesadas de Miosina/genética , RNA Mensageiro/genética , Acetatos/isolamento & purificação , Acetilcisteína/farmacologia , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/antagonistas & inibidores , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Catecóis/isolamento & purificação , Diferenciação Celular , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/genética , Complexo II de Transporte de Elétrons/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Azeite de Oliva , Estresse Oxidativo , Óleos de Plantas/química , Proteólise , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , terc-Butil Hidroperóxido/antagonistas & inibidores , terc-Butil Hidroperóxido/farmacologiaRESUMO
A biological microelectromechanical system (BioMEMS) device was designed to study complementary mitochondrial parameters important in mitochondrial dysfunction studies. Mitochondrial dysfunction has been linked to many diseases, including diabetes, obesity, heart failure and aging, as these organelles play a critical role in energy generation, cell signaling and apoptosis. The synthesis of ATP is driven by the electrical potential across the inner mitochondrial membrane and by the pH difference due to proton flux across it. We have developed a tool to study the ionic activity of the mitochondria in parallel with dielectric measurements (impedance spectroscopy) to gain a better understanding of the properties of the mitochondrial membrane. This BioMEMS chip includes: 1) electrodes for impedance studies of mitochondria designed as two- and four-probe structures for optimized operation over a wide frequency range and 2) ion-sensitive field effect transistors for proton studies of the electron transport chain and for possible monitoring other ions such as sodium, potassium and calcium. We have used uncouplers to depolarize the mitochondrial membrane and disrupt the ionic balance. Dielectric spectroscopy responded with a corresponding increase in impedance values pointing at changes in mitochondrial membrane potential. An electrical model was used to describe mitochondrial sample's complex impedance frequency dependencies and the contribution of the membrane to overall impedance changes. The results prove that dielectric spectroscopy can be used as a tool for membrane potential studies. It can be concluded that studies of the electrochemical parameters associated with mitochondrial bioenergetics may render significant information on various abnormalities attributable to these organelles.
Assuntos
Espectroscopia Dielétrica , Fenômenos Mecânicos , Microtecnologia/instrumentação , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Eletrodos , Glutamatos/farmacologia , Concentração de Íons de Hidrogênio , Malatos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Membranas Mitocondriais/efeitos dos fármacos , Transistores EletrônicosRESUMO
Obesity is a primary risk factor for numerous metabolic diseases including metabolic syndrome, type II diabetes (T2DM), cardiovascular disease and cancer. Although classically viewed as a storage organ, the field of white adipose tissue biology is expanding to include the consideration of the tissue as an endocrine organ and major contributor to overall metabolism. Given its role in energy production, the mitochondrion has long been a focus of study in metabolic dysfunction and a link between the organelle and white adipose tissue function is likely. Herein, we present a novel method for assessing mitochondrial bioenergetics from whole white adipose tissue. This method requires minimal manipulation of tissue, and eliminates the need for cell isolation and culture. Additionally, this method overcomes some of the limitations to working with transformed and/or isolated primary cells and allows for results to be obtained more expediently. In addition to the novel method, we present a comprehensive statistical analysis of bioenergetic data as well as guidelines for outlier analysis.
Assuntos
Tecido Adiposo Branco/metabolismo , Mitocôndrias/metabolismo , Animais , Antibacterianos/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Metabolismo Energético/efeitos dos fármacos , Masculino , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Rotenona/farmacologiaRESUMO
Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1α in response to 1% O2 hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that altered mitochondrial fission dynamics represents a phenotype of a subpopulation of EOCs.
Assuntos
Glicólise , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Neoplasias Ovarianas/metabolismo , Consumo de Oxigênio , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mitocôndrias/patologia , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/patologia , Ionóforos de Próton/farmacologiaRESUMO
In vitro and in vivo models of Parkinson's disease (PD) suggest that increased oxidant production leads to mitochondrial dysfunction in dopaminergic neurons and subsequent cell death. However, it remains unclear if cell death in these models is caused by inhibition of mitochondrial function or oxidant production. The objective of this study was to determine the relationship between mitochondrial dysfunction and oxidant production in response to multiple PD neurotoxicant mimetics. MPP(+) caused a dose-dependent decrease in the basal oxygen consumption rate in dopaminergic N27 cells, indicating a loss of mitochondrial function. In parallel, we found that MPP(+) only modestly increased oxidation of hydroethidine as a diagnostic marker of superoxide production in these cells. Similar results were found using rotenone as a mitochondrial inhibitor, or 6-hydroxydopamine (6-OHDA) as a mechanistically distinct PD neurotoxicant, but not with exposure to paraquat. In addition, the extracellular acidification rate, used as a marker of glycolysis, was stimulated to compensate for oxygen consumption rate inhibition after exposure to MPP(+), rotenone, or 6-OHDA, but not paraquat. Together these data indicate that MPP(+), rotenone, and 6-OHDA dramatically shift bioenergetic function away from the mitochondria and towards glycolysis in N27 cells.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Metabolismo Energético/efeitos dos fármacos , Neurotoxinas/farmacologia , Superóxidos/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Trifosfato de Adenosina/metabolismo , Adrenérgicos/farmacologia , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Linhagem Celular Transformada , Dopaminérgicos/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Herbicidas/farmacologia , Inseticidas/farmacologia , Oligomicinas/farmacologia , Oxidopamina/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Paraquat/farmacologia , Ionóforos de Próton/farmacologia , Ratos , Rotenona/farmacologia , Fatores de TempoRESUMO
The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and therefore masked by, alternative pathways which have significant spare capacity. Here we present a multiplexed 'cell energy budget' platform which facilitates metabolic assessment and cross-comparison of different cells and the identification of genes directly or indirectly involved in ATP production. Long-decay emitting O(2) and pH sensitive probes and time-resolved fluorometry are used to measure changes in cellular O(2) consumption, glycolytic and total extracellular acidification (ECA), along with the measurement of total ATP and protein content in multiple samples. To assess the extent of spare capacity in the main energy pathways, the cells are also analysed following double-treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and oligomycin. The four-parametric platform operating in a high throughput format has been validated with two panels of transformed cells: mouse embryonic fibroblasts (MEFs) lacking the Krebs cycle enzyme fumarate hydratase (Fh1) and HeLa cells with reduced expression of pyrimidine nucleotide carrier 1. In both cases, a marked reduction in both respiration and spare respiratory capacity was observed, accompanied by a compensatory activation of glycolysis and consequent maintenance of total ATP levels. At the same time, in Fh1-deficient MEFs the contribution of non-glycolytic pathways to the ECA did not change.
Assuntos
Metabolismo Energético/fisiologia , Técnicas de Inativação de Genes , Interferência de RNA/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Dióxido de Carbono/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Ciclo do Ácido Cítrico/fisiologia , Embrião de Mamíferos/citologia , Metabolismo Energético/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Fibroblastos/metabolismo , Fumarato Hidratase/deficiência , Fumarato Hidratase/genética , Deleção de Genes , Glicólise/fisiologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Ácido Láctico/metabolismo , Camundongos , Proteínas de Transporte da Membrana Mitocondrial , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Proteínas de Transporte de Nucleotídeos/genética , Oligomicinas/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , RNA Interferente Pequeno/genéticaRESUMO
Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Here, we evaluated the in vitro effects of FCCP on the growth of Calu-6 lung cancer cells. FCCP inhibited the growth of Calu-6 cells with an IC(50) of approximately 6.64+/-1.84 microM at 72 h, as shown by MTT. DNA flow cytometric analysis indicated that FCCP induced G1 phase arrest below 20 microM of FCCP. Treatment with FCCP decreased the level of CDKs and cyclines in relation to G1 phase. In addition, FCCP not only increased the p27 level but also enhanced its binding with CDK4, which was associated with hypophosphorylation of Rb protein. While transfection of p27 siRNA inhibited G1 phase arrest in FCCP-treated cells, it did not enhance Rb phosphorylation. FCCP also efficiently induced apoptosis. The apoptotic process was accompanied with an increase in sub-G1 cells, annexin V staining cells, mitochondria membrane potential (MMP) loss and cleavage of PARP protein. All of the caspase inhibitors (caspase-3, -8, -9 and pan-caspase inhibitor) markedly rescued the Calu-6 cells from FCCP-induced cell death. However, knock down of p27 protein intensified FCCP-induced cell death. Moreover, FCCP induced the depletion of GSH content in Calu-6 cells, which was prevented by all of the caspase inhibitors. In summary, our results demonstrated that FCCP inhibits the growth of Calu-6 cells in vitro. The growth inhibitory effect of FCCP might be mediated by cell cycle arrest and apoptosis via decrease of CDKs and caspase activation, respectively. These findings now provide a better elucidation of the mechanisms involved in FCCP-induced growth inhibition in lung cancer.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Desacopladores/farmacologia , Adenocarcinoma/patologia , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , DNA de Neoplasias/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Formazans/metabolismo , Fase G1/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Sais de Tetrazólio/metabolismo , Fatores de TempoRESUMO
Indirubin, a red colored 3,2'-bisindole isomer, is a component of Indigo naturalis and is an active ingredient used in traditional Chinese medicine for the treatment of chronic diseases. The family of indirubin derivatives, such as indirubin-3'-oxime, has been suggested for various therapeutic indications. However, potential toxic interactions such as indirubin effects on mitochondrial bioenergetics are still unknown. This study evaluated the action of indirubin-3'-oxime on the function of isolated rat liver mitochondria contributing to a better understanding of the biochemical mechanisms underlying the multiple effects of indirubin. Indirubin-3'-oxime incubated with isolated rat liver mitochondria, at concentrations above 10microM, significantly depresses the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial membrane potential and in the phosphorylative cycle induced by ADP. Furthermore, indirubin-3'-oxime at up to 25microM stimulates the rate of state 4 respiration and inhibits state 3 respiration. The increased lag phase of repolarization was associated with a direct inhibition of the mitochondrial ATPase. Indirubin-3'-oxime significantly inhibited the activity of complex II and IV thus explaining the decreased FCCP-stimulated mitochondrial respiration. Mitochondria pre-incubated with indirubin-3'-oxime exhibits decreased susceptibility to calcium-induced mitochondrial permeability transition. This work shows for the first time multiple effects of indirubin-3'-oxime on mitochondrial bioenergetics thus indicating a potential mechanism for indirubin-3'-oxime effects on cell function.
Assuntos
Indóis/toxicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Oximas/toxicidade , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Animais , Cálcio/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Respiração Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/química , Indóis/administração & dosagem , Masculino , Mitocôndrias Hepáticas/metabolismo , Oximas/administração & dosagem , Oxigênio/metabolismo , Permeabilidade/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O2(-)). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O2(-) generation, besides complex III, stimulated NO degradation. Larger amounts of O2(-) were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O2(-) production were stimulated by free Ca2+ concentration. Together, these results indicate that Ca2+-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O2(-) production that favors NO degradation in potato tuber mitochondria.
Assuntos
Mitocôndrias/metabolismo , NADH Desidrogenase/metabolismo , Óxido Nítrico/metabolismo , Solanum tuberosum/metabolismo , Cálcio/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Peróxido de Hidrogênio/metabolismo , Malatos/farmacologia , Mitocôndrias/efeitos dos fármacos , NADP/farmacologia , Rotenona/farmacologia , Ácido Succínico/farmacologia , Superóxidos/metabolismo , Desacopladores/farmacologiaRESUMO
We investigated the mechanism by which 3,5-diiodo-l-thyronine (T2) affects skeletal muscle mitochondrial bioenergetic parameters following its acute administration to hypothyroid rats. One hour after injection, T2 increased both coupled and uncoupled respiration rates by +27% and +42%, respectively. Top-down elasticity analysis revealed that these effects were the result of increases in the substrate oxidation and mitochondrial uncoupling. Discriminating between proton-leak and redox-slip processes, we identified an increased mitochondrial proton conductance as the "pathway" underlying the effect of T2 on mitochondrial uncoupling. As a whole, these results may provide a mechanism by which T2 rapidly affects energy metabolism in hypothyroid rats.
Assuntos
Di-Iodotironinas/administração & dosagem , Di-Iodotironinas/farmacologia , Metabolismo Energético/efeitos dos fármacos , Hipotireoidismo/metabolismo , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Respiração Celular/efeitos dos fármacos , Cinética , Masculino , Malonatos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Oligomicinas/farmacologia , Oxirredução/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Prótons , Ratos , Ratos WistarRESUMO
Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a)quinolizinium] is an alkaloid present in plant extracts and has a history of use in traditional Chinese and Native American medicine. Because of its ability to arrest the cell cycle and cause apoptosis of several malignant cell lines, it has received attention as a potential anticancer therapeutic agent. Previous studies suggest that mitochondria may be an important target of berberine, but relatively little is known about the extent or molecular mechanisms of berberine-mitochondrial interactions. The objective of the present work was to investigate the interaction of berberine with mitochondria, both in situ and in isolated mitochondrial fractions. The data show that berberine is selectively accumulated by mitochondria, which is accompanied by arrest of cell proliferation, mitochondrial fragmentation and depolarization, oxidative stress, and a decrease in ATP levels. Electron microscopy of berberine-treated cells shows a reduction in mitochondria-like structures, accompanied by a decrease in mitochondrial DNA copy number. Isolated mitochondrial fractions treated with berberine had slower mitochondrial respiration, especially when complex I substrates were used, and increased complex I-dependent oxidative stress. It is also demonstrated for the first time that berberine stimulates the mitochondrial permeability transition. Direct effects on ATPase activity were not detected. The present work demonstrates a number of previously unknown alterations of mitochondrial physiology induced by berberine, a potential chemotherapeutic agent, although it also suggests that high doses of berberine should not be used without a proper toxicology assessment.
Assuntos
Berberina/farmacologia , Mitocôndrias/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Animais , Berberina/farmacocinética , Cálcio/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , DNA Mitocondrial/análise , Metabolismo Energético/efeitos dos fármacos , Masculino , Melanoma Experimental/patologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Intracellular Ca2+ signaling controls many cellular functions. Understanding its regulation by selenoproteins is essential for understanding the role of selenoproteins in regulating cell functions. The activity of thioredoxin reductase (TrxR), thioredoxin (Trx) content, and the activity of glutathione peroxidase (GPx) in the human endothelial cells cultured in selenium-supplemented medium (refer as Se+ cells) was found 70%, 40%, and 20% higher, respectively than those in the cells cultured in normal medium (refer as Se0 cells). The intracellular Ca2+ signaling initiated by inositol 1,4,5-trisphosphate (IP3), histamine, thapsigargin (TG), carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone (FCCP), and cyclosporin A (CsA) was investigated in both Se+ and Se0 cells. It was interestingly found that the higher activity of selenoproteins reduced the sensitivity of IP3 receptor to the IP3-triggered Ca2+ release from intracellular stores, but enhanced activation of the receptor-coupled phospholipase C in histamine-stimulated Se+ cells by showing much more generation of IP3 and higher elevation of cytosolic Ca2+. The higher selenoprotein activity also reduced susceptibility of the uniporter to the mitochondrial uncoupler, susceptibility of the permeability transition pore (PTP) to its inhibitor, and the vulnerability of endoplasmic reticulum (ER) Ca2+-ATPase to its inhibitor in selenium-supplementing cells. The results suggest that cell calcium signaling is subjected to thiol-redox regulation by selenoproteins.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Selênio/farmacologia , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ciclosporina/farmacologia , Glutationa Peroxidase/metabolismo , Histamina/farmacologia , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Triglicerídeos/metabolismoRESUMO
The mitochondrion plays a pivotal role in energy metabolism in eukaryotic cells. The electrochemical potential across the mitochondrial inner membrane is regulated to cope with cellular energy needs and thus reflects the bioenergetic state of the cell. Traditional assays for mitochondrial membrane potential are not amenable to high-throughput drug screening. In this paper, I describe a high-throughput assay that measures the mitochondrial membrane potential of living cells in 96- or 384-well plates. Cells were first treated with test compounds and then with a fluorescent potentiometric probe, the cationic-lipophilic dye tetramethylrhodamine methyl ester (TMRM). The cells were then washed to remove free compounds and probe. The amount of TMRM retained in the mitochondria, which is proportional to the mitochondrial membrane potential, was measured on an LJL Analyst fluorescence reader. Under optimal conditions, the assay measured only the mitochondrial membrane potential. The chemical uncouplers carbonylcyanide m-chlorophenyl hydrazone and dinitrophenol decreased fluorescence intensity, with IC(50) values (concentration at 50% inhibition) similar to those reported in the literature. A Z' factor of greater than 0.5 suggests that this cell-based assay can be adapted for high-throughput screening of chemical libraries. This assay may be used in screens for drugs to treat metabolic disorders such as obesity and diabetes, as well as cancer and neurodegenerative diseases.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Membranas Intracelulares/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Bioensaio/métodos , Células CHO , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Permeabilidade da Membrana Celular , Cricetinae , Dinitrofenóis/farmacologia , Corantes Fluorescentes , Fluorometria/métodos , Humanos , Membranas Intracelulares/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/fisiologia , Rodaminas/farmacologia , Células Tumorais CultivadasRESUMO
Inhibition of inward rectifier K(+) channels under ischemic conditions may contribute to electrophysiological consequences of ischemia such as cardiac arrhythmia. Ischemia causes metabolic inhibition, and the use of metabolic inhibitors is one experimental method of simulating ischemia. The effects of metabolic inhibitors on the activity of inward rectifier K(+) channels K(ir)2.1, K(ir)2.2, and K(ir)2.3 were studied by heterologous expression in Xenopus oocytes and two-electrode voltage clamp. 10 microm carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) inhibited K(ir)2.2 and K(ir)2.3 currents but was without effect on K(ir)2.1 currents. The rate of decline of current in FCCP was faster for K(ir)2.3 than for K(ir)2.2. K(ir)2.3 was inhibited by 3 mm sodium azide (NaN(3)), whereas K(ir)2.1 and K(ir)2.2 were not. K(ir)2.2 was inhibited by 10 mm NaN(3). All three of these inward rectifiers were inhibited by lowering the pH of the solution perfusing inside-out membrane patches. K(ir)2.3 was most sensitive to pH (pK = 6.9), whereas K(ir)2.1 was least sensitive (pK = 5.9). For K(ir)2.2 the pK was 6.2. These results demonstrate the differential sensitivity of these inward rectifiers to metabolic inhibition and internal pH. The electrophysiological response of a particular cell type to ischemia may depend on the relative expression levels of different inward rectifier genes.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização/farmacologia , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Clonagem Molecular , DNA Complementar/metabolismo , Concentração de Íons de Hidrogênio , Ionóforos/farmacologia , Potenciais da Membrana , Oócitos/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , Azida Sódica/farmacologia , Transcrição Gênica , Desacopladores/farmacologia , Xenopus laevisRESUMO
In cardiomyocytes sarcolemmal KATP channels open massively when the cytosolic [ATP] drops into the range of tens of micromolar, as during acute ischemia. The diuretic drug amiloride and related derivatives are well established as drugs blocking the Na+/H+- and the Na+/Ca2+-exchange, protecting the ischemic heart. Herein, the blocking action of amiloride and its derivatives 2',4'-dichlorobenzamil (DCB) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) on KATP channels was tested. In inside-out patches of mouse cardiac myocytes, amiloride, DCB, and EIPA reversibly blocked the KATP channels with the IC50 values 102, 1.80, and 2.14 micromol/l (-80 mV), respectively. Similar IC50 values were obtained in recombinant channels when coexpressing the KIR6.2 subunit with one of the sulfonylurea receptors SUR1 and SUR2A. All three drugs also blocked currents generated by the C-terminus deletion mutant KIR6.2delta26 in the absence of SUR. Amiloride blocked outward currents more effectively than inward currents whereas the block by DCB and EIPA was voltage independent. In cardiomyocytes, also whole-cell IKATP was blocked by the three drugs. In conclusion, amiloride, EIPA, and DCB block the pore-forming KIR6.2 subunit of cardiac KATP channels with higher potency than the Na+/H+- and the Na+/Ca2+-exchange, precluding a specific block of the exchanges under ischemic conditions.
Assuntos
Amilorida/análogos & derivados , Amilorida/farmacologia , Bloqueadores dos Canais de Potássio , Canais de Potássio , Transportadores de Cassetes de Ligação de ATP , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Eletrofisiologia , Coração/efeitos dos fármacos , Canais KATP , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Miocárdio/citologia , Miocárdio/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Plasmídeos/genética , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , RNA Complementar/biossíntese , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Desacopladores/farmacologia , XenopusRESUMO
Ethylazinphos increases the passive proton permeability of lipid bilayers reconstituted with dipalmitoylphosphatidylcholine (DPPC) and mitochondrial lipids. A sharp increase of proton permeability is detected at insecticide/lipid molar ratios identical to those inducing phase separation in the plane of DPPC bilayers, as revealed by differential scanning calorimetry (DSC). Ethylazinphos progressively depresses the transmembrane potential (DeltaPsi) of mitochondria supported by piruvate/malate, succinate, or ascorbate/TMPD. Additionally, a decreased depolarization induced by ADP depends on ethylazinphos concentration, reflecting a phosphorylation depression. This loss of phosphorylation is a consequence of a decreased DeltaPsi. A decreased respiratory control ratio is also observed, since ethylazinphos stimulates state 4 respiration and inhibits ADP-stimulated respiration (state 3). Ethylazinphos concentrations up to 100 nmol/mg mitochondrial protein increase the rate of state 4 together with a decrease in DeltaPsi, without significant perturbation of state 3 and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP)-uncoupled respiration. For increased insecticide concentrations, the state 3 and FCCP-uncoupled respiration are inhibited to approximately the same extent. The perturbations are more pronounced when the energization is supported by pyruvate/malate and less effective when succinate is used as substrate. The present data, in association with previous DSC studies, indicate that ethylazinphos, at concentrations up to 100 nmol/mg mitochondrial protein, interacts with the lipid bilayer of mitochondrial membrane, changing the lipid organization and increasing the proton permeability of the inner membrane. The increased proton permeability explains the decreased oxidative phosphorylation coupling. Resulting disturbed ATP synthesis may significantly underlie the mechanisms of ethylazinphos toxicity, since most of cell energy in eukaryotes is provided by mitochondria.
Assuntos
Azinfos-Metil/farmacologia , Inseticidas/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Prótons , Animais , Azinfos-Metil/análogos & derivados , Varredura Diferencial de Calorimetria , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/fisiologia , Relação Dose-Resposta a Droga , Feminino , Masculino , Lipídeos de Membrana/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mitocôndrias Hepáticas/metabolismo , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Ring-B derivatization of totarol (1) afforded the series of compounds 2-22 which were screened in vitro against: beta-lactamase-positive and high level gentamycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), and multiresistant Klebsiella pneumoniae. Several of the derivatives retained much of the antibacterial activity of totatol against the first three of these organisms (all gram-positive), but none was more active. The gram-negative Klebsiella was resistant to all compounds examined. Totarol (1) was shown to uncouple oxidative phosphorylation in isolated mitochondria at 50 microM.