RESUMEN
Understanding the role of astrocytes in the development of the nervous system and neurodegenerative disorders implies a necessary knowledge of the oxidative metabolism of proliferating astrocytes. The electron flux through mitochondrial respiratory complexes and oxidative phosphorylation may impact the growth and viability of these astrocytes. Here, we aimed at assessing to which extent mitochondrial oxidative metabolism is required for astrocyte survival and proliferation. Primary astrocytes from the neonatal mouse cortex were cultured in a physiologically relevant medium with the addition of piericidin A or oligomycin at concentrations that fully inhibit complex I-linked respiration and ATP synthase, respectively. The presence of these mitochondrial inhibitors for up to 6 days in a culture medium elicited only minor effects on astrocyte growth. Moreover, neither the morphology nor the proportion of glial fibrillary acidic protein-positive astrocytes in culture was affected by piericidin A or oligomycin. Metabolic characterization of the astrocytes showed a relevant glycolytic metabolism under basal conditions, despite functional oxidative phosphorylation and large spare respiratory capacity. Our data suggest that astrocytes in primary culture can sustainably proliferate when their energy metabolism relies only on aerobic glycolysis since their growth and survival do not require electron flux through respiratory complex I or oxidative phosphorylation.
Asunto(s)
Complejo I de Transporte de Electrón , Fosforilación Oxidativa , Ratones , Animales , Complejo I de Transporte de Electrón/metabolismo , Astrocitos/metabolismo , Mitocondrias/metabolismo , Oligomicinas/farmacologíaRESUMEN
Microcystin-LR (MC-LR) is a potent cyanotoxin that can reach several organs. However subacute exposure to sublethal doses of MC-LR has not yet well been studied. Herein, we evaluated the outcomes of subacute and sublethal MC-LR exposure on lungs. Male BALB/c mice were exposed to MC-LR by gavage (30 µg/kg) for 20 consecutive days, whereas CTRL mice received filtered water. Respiratory mechanics was not altered in MC-LR group, but histopathology disclosed increased collagen deposition, immunological cell infiltration, and higher percentage of collapsed alveoli. Mitochondrial function was extensively affected in MC-LR animals. Additionally, a direct in vitro titration of MC-LR revealed impaired mitochondrial function. In conclusion, MC-LR presented an intense deleterious effect on lung mitochondrial function and histology. Furthermore, MC-LR seems to exert an oligomycin-like effect in lung mitochondria. This study opens new perspectives for the understanding of the putative pulmonary initial mechanisms of damage resulting from oral MC-LR intoxication.
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Microcistinas , Mitocondrias , Animales , Ingestión de Alimentos , Pulmón , Masculino , Toxinas Marinas , Ratones , Microcistinas/metabolismo , Microcistinas/toxicidad , Oligomicinas/metabolismo , Oligomicinas/farmacologíaRESUMEN
Objectives: Mesenchymal Stem/Stromal Cells (MSC) are promising therapeutic tools for inflammatory diseases due to their potent immunoregulatory capacities. Their suppressive activity mainly depends on inflammatory cues that have been recently associated with changes in MSC bioenergetic status towards a glycolytic metabolism. However, the molecular mechanisms behind this metabolic reprogramming and its impact on MSC therapeutic properties have not been investigated. Methods: Human and murine-derived MSC were metabolically reprogramed using pro-inflammatory cytokines, an inhibitor of ATP synthase (oligomycin), or 2-deoxy-D-glucose (2DG). The immunosuppressive activity of these cells was tested in vitro using co-culture experiments with pro-inflammatory T cells and in vivo with the Delayed-Type Hypersensitivity (DTH) and the Graph versus Host Disease (GVHD) murine models. Results: We found that the oligomycin-mediated pro-glycolytic switch of MSC significantly enhanced their immunosuppressive properties in vitro. Conversely, glycolysis inhibition using 2DG significantly reduced MSC immunoregulatory effects. Moreover, in vivo, MSC glycolytic reprogramming significantly increased their therapeutic benefit in the DTH and GVHD mouse models. Finally, we demonstrated that the MSC glycolytic switch effect partly depends on the activation of the AMPK signaling pathway. Conclusion: Altogether, our findings show that AMPK-dependent glycolytic reprogramming of MSC using an ATP synthase inhibitor contributes to their immunosuppressive and therapeutic functions, and suggest that pro-glycolytic drugs might be used to improve MSC-based therapy.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Glucólisis/efectos de los fármacos , Enfermedad Injerto contra Huésped/inmunología , Hipersensibilidad Tardía/inmunología , Células Madre Mesenquimatosas/efectos de los fármacos , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Animales , Antimetabolitos/farmacología , Linfocitos T CD4-Positivos , Desoxiglucosa/farmacología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Humanos , Inmunoterapia , Ácido Láctico/metabolismo , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/metabolismo , Ratones , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Oligomicinas/farmacología , Fosforilación Oxidativa , Consumo de OxígenoRESUMEN
Mesenchymal stem cell (MSC)-based therapy is being increasingly considered a powerful opportunity for several disorders based on MSC immunoregulatory properties. Nonetheless, MSC are versatile and plastic cells that require an efficient control of their features and functions for their optimal use in clinic. Recently, we have shown that PPARß/δ is pivotal for MSC immunoregulatory and therapeutic functions. However, the role of PPARß/δ on MSC metabolic activity and the relevance of PPARß/δ metabolic control on MSC immunosuppressive properties have never been addressed. Here, we demonstrate that PPARß/δ deficiency forces MSC metabolic adaptation increasing their glycolytic activity required for their immunoregulatory functions on Th1 and Th17 cells. Additionally, we show that the inhibition of the mitochondrial production of ATP in MSC expressing PPARß/δ, promotes their metabolic switch towards aerobic glycolysis to stably enhance their immunosuppressive capacities significantly. Altogether, these data demonstrate that PPARß/δ governs the immunoregulatory potential of MSC by dictating their metabolic reprogramming and pave the way for enhancing MSC immunoregulatory properties and counteracting their versatility.
Asunto(s)
Células Madre Mesenquimatosas/metabolismo , PPAR-beta/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Células de la Médula Ósea/citología , Linfocitos T CD4-Positivos/citología , Proliferación Celular , Silenciador del Gen , Glucólisis , Terapia de Inmunosupresión , Ratones , Oligomicinas/química , Células TH1/citología , Células Th17/citologíaRESUMEN
This study sought to elucidate how oligomycin, an ATP synthase blocker, leads to underestimation of maximal oxygen consumption rate (maxOCR) and spare respiratory capacity (SRC) in tumor cells. T98G and U-87MG glioma cells were titrated with the protonophore CCCP to induce maxOCR. The presence of oligomycin (0.3-3.0 µg/mL) led to underestimation of maxOCR and a consequent decrease in SRC values of between 25% and 40% in medium containing 5.5 or 11 mM glucose. The inhibitory effect of oligomycin on CCCP-induced maxOCR did not occur when glutamine was the metabolic substrate or when the glycolytic inhibitor 2-deoxyglucose was present. ATP levels were reduced and ADP/ATP ratios increased in cells treated with CCCP, but these changes were minimized when oligomycin was used to inhibit reverse activity of ATP synthase. Exposing digitonin-permeabilized cells to exogenous ATP, but not ADP, resulted in partial inhibition of CCCP-induced maxOCR. We conclude that underestimation of maxOCR and SRC in tumor cells when ATP synthase is inhibited is associated with high glycolytic activity and that the glycolytic ATP yield may have an inhibitory effect on the metabolism of respiratory substrates and cytochrome c oxidase activity. Under CCCP-induced maxOCR, oligomycin preserves intracellular ATP by inhibiting ATP synthase reverse activity.
Asunto(s)
Adenosina Trifosfato/metabolismo , Transporte de Electrón/fisiología , Glucólisis/fisiología , Mitocondrias/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Neoplasias/metabolismo , Línea Celular Tumoral , Desoxiglucosa/metabolismo , Humanos , Oligomicinas/metabolismo , Consumo de Oxígeno/fisiologíaRESUMEN
Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2.
Asunto(s)
Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/fisiología , Mitocondrias/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Humanos , Interferón gamma/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/efectos de los fármacos , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Oligomicinas/farmacología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genéticaRESUMEN
The maximal capacity of the mitochondrial electron transport system (ETS) in intact cells is frequently estimated by promoting protonophore-induced maximal oxygen consumption preceded by inhibition of oxidative phosphorylation by oligomycin. In the present study, human glioma (T98G and U-87MG) and prostate cancer (PC-3) cells were titrated with different concentrations of the protonophore CCCP to induce maximal oxygen consumption rate (OCR) within respirometers in a conventional growth medium. The results demonstrate that the presence of oligomycin or its A-isomer leads to underestimation of maximal ETS capacity. In the presence of oligomycin, the spare respiratory capacity (SRC), i.e., the difference between the maximal and basal cellular OCR, was underestimated by 25 to 45%. The inhibitory effect of oligomycin on SRC was more pronounced in T98G cells and was observed in both suspended and attached cells. Underestimation of SRC also occurred when oxidative phosphorylation was fully inhibited by the ATP synthase inhibitor citreoviridin. Further experiments indicated that oligomycin cannot be replaced by the adenine nucleotide translocase inhibitors bongkrekic acid or carboxyatractyloside because, although these compounds have effects in permeabilized cells, they do not inhibit oxidative phosphorylation in intact cells. We replaced CCCP by FCCP, another potent protonophore and similar results were observed. Lower maximal OCR and SRC values were obtained with the weaker protonophore 2,4-dinitrophenol, and these parameters were not affected by the presence of oligomycin. In permeabilized cells or isolated brain mitochondria incubated with respiratory substrates, only a minor inhibitory effect of oligomycin on CCCP-induced maximal OCR was observed. We conclude that unless a previously validated protocol is employed, maximal ETS capacity in intact cells should be estimated without oligomycin. The inhibitory effect of an ATP synthase blocker on potent protonophore-induced maximal OCR may be associated with impaired metabolism of mitochondrial respiratory substrates.
Asunto(s)
Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oligomicinas/farmacología , Línea Celular Tumoral , Respiración de la Célula/efectos de los fármacos , Transporte de Electrón/efectos de los fármacos , Humanos , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacosRESUMEN
The transition from primary to secondary metabolism in antibiotic-producing Streptomyces correlates with expression of genes involved in stress responses. Consequently, regulatory pathways that regulate specific stress responses are potential targets to manipulate to increase antibiotic titers. In this study, genes encoding key proteins involved in regulation of the osmotic stress response in Streptomyces avermitilis, the industrial producer of avermectins, are investigated as targets. Disruption of either osaBSa, encoding a response regulator protein, or osaCSa, encoding a multidomain regulator of the alternative sigma factor SigB, led to increased production of both oligomycin, by up to 200%, and avermectin, by up to 37%. The mutations also conditionally affected morphological development; under osmotic stress, the mutants were unable to erect an aerial mycelium. In addition, we demonstrate the delivery of DNA into a streptomycete using biolistics. The data reveal that information on stress regulatory responses can be integrated in rational strain improvement to improve yields of bioactive secondary metabolites.
Asunto(s)
Antibacterianos/metabolismo , Osmorregulación , Streptomyces/genética , Streptomyces/metabolismo , Eliminación de Gen , Redes Reguladoras de Genes , Ivermectina/análogos & derivados , Ivermectina/metabolismo , Ingeniería Metabólica , Oligomicinas/metabolismo , Streptomyces/fisiologíaRESUMEN
The F1FO-ATP synthase of the colorless alga Polytomella sp. exhibits a robust peripheral arm constituted by nine atypical subunits only present in chlorophycean algae. The isolated dimeric enzyme exhibits a latent ATP hydrolytic activity which can be activated by some detergents. To date, the kinetic behavior of the algal ATPase has not been studied. Here we show that while the soluble F1 sector exhibits Michaelis-Menten kinetics, the dimer exhibits a more complex behavior. The kinetic parameters (Vmax and Km) were obtained for both the F1 sector and the dimeric enzyme as isolated or activated by detergent, and this activation was also seen on the enzyme reconstituted in liposomes. Unlike other ATP synthases, the algal dimer hydrolyzes ATP on a wide range of pH and temperature. The enzyme was inhibited by oligomycin, DCCD and Mg-ADP, although oligomycin induced a peculiar inhibition pattern that can be attributed to structural differences in the algal subunit-c. The hydrolytic activity was temperature-dependent and exhibited activation energy of 4 kcal/mol. The enzyme also exhibited a hysteretic behavior with a lag phase strongly dependent on temperature but not on pH, that may be related to a possible regulatory role in vivo.
Asunto(s)
Adenosina Trifosfato/metabolismo , ATPasas de Translocación de Protón/metabolismo , Volvocida/enzimología , Adenosina Difosfato/farmacología , Diciclohexilcarbodiimida/farmacología , Dimerización , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Cinética , Oligomicinas/farmacología , Proteolisis , ATPasas de Translocación de Protón/antagonistas & inhibidoresRESUMEN
Gamma aminobutyric acid (GABA) is widely known as a neurotransmitter and signal transduction molecule found in vertebrates, plants, and some protozoan organisms. However, the presence of GABA and its role in trypanosomatids is unknown. Here, we report the presence of intracellular GABA and the biochemical characterization of its uptake in Trypanosoma cruzi, the etiological agent of Chagas' disease. Kinetic parameters indicated that GABA is taken up by a single transport system in pathogenic and nonpathogenic forms. Temperature dependence assays showed a profile similar to glutamate transport, but the effect of extracellular cations Na(+) , K(+) , and H(+) on GABA uptake differed, suggesting a different uptake mechanism. In contrast to reports for other amino acid transporters in T. cruzi, GABA uptake was Na(+) dependent and increased with pH, with a maximum activity at pH 8.5. The sensitivity to oligomycin showed that GABA uptake is dependent on ATP synthesis. These data point to a secondary active Na(+) /GABA symporter energized by Na(+) -exporting ATPase. Finally, we show that GABA occurs in the parasite's cytoplasm under normal culture conditions, indicating that it is regularly taken up from the culture medium or synthesized through an still undescribed metabolic pathway.
Asunto(s)
Trypanosoma cruzi/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Adenosina Trifosfato/biosíntesis , Aminoácidos/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Ácido Glutámico/metabolismo , Concentración de Iones de Hidrógeno , Oligomicinas/farmacología , Potasio/metabolismo , Sodio/metabolismo , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/ultraestructuraRESUMEN
Because mitochondrial oxidative stress and impairment are important mediators of neuronal damage in neurodegenerative diseases and in brain ischemia/reperfusion, in the present study, we evaluated the antioxidant and mitoprotective effect of a new promising neuroprotective molecule, JM-20, in mitochondria and synaptosomes isolated from rat brains. JM-20 inhibited succinate-mediated H2O2 generation in both mitochondria and synaptosomes incubated in depolarized (high K(+)) medium at extremely low micromolar concentration and with identical IC50 values of 0.91 µM. JM-20 also repressed glucose-induced H2O2 generation stimulated by rotenone or by antimycin A in synaptosomes incubated in high sodium-polarized medium at extremely low IC50 values of 0.395 µM and 2.452 µM, respectively. JM-20 was unable to react directly with H2O2 or with superoxide anion radicals but displayed a cathodic reduction peak at -0.71V, which is close to that of oxygen (-0.8V), indicating high electron affinity. JM-20 also inhibited uncoupled respiration in mitochondria or synaptosomes and was a more effective inhibitor in the presence of the respiratory substrates glutamate/malate than in the presence of succinate. JM-20 also prevented Ca(2+)-induced mitochondrial permeability transition pore opening, membrane potential dissipation and cytochrome c release, which are key pathogenic events during stroke. This molecule also prevented Ca(2+) influx into synaptosomes and mitochondria; the former effect was a consequence of the latter because JM-20 inhibition followed the patterns of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), which is a classic mitochondrial uncoupler. Because the mitochondrion is considered an important source and target of neuronal cell death signaling after an ischemic insult, the antioxidant and protective effects of JM-20 against the deleterious effects of Ca(2+) observed at the mitochondrial level in this study may endow this molecule with the ability to succeed in mitochondrion-targeted strategies to combat ischemic brain damage.
Asunto(s)
Antioxidantes/farmacología , Benzodiazepinas/farmacología , Calcio/toxicidad , Mitocondrias/efectos de los fármacos , Niacina/análogos & derivados , Prosencéfalo/ultraestructura , Sinaptosomas/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Catalasa/farmacología , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Niacina/farmacología , Oligomicinas/farmacología , Oxígeno/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismoRESUMEN
Energy metabolism in the adult brain consumes large quantities of glucose, but little is known to date regarding how glucose metabolism changes during neuronal differentiation, a process that is highly demanding energetically. We studied changes in glucose metabolism during neuronal differentiation of P19 mouse embryonal carcinoma cells, E14Tg2A embryonic stem cells as well as during brain development of BLC57 mice. In all these models, we find that neurogenesis is accompanied by a shift from oxidative to fermentative glucose metabolism. This shift is accompanied by both a decrease in mitochondrial enzymatic activities and mitochondrial uncoupling. In keeping with this finding, we also observe that differentiation does not require oxidative metabolism, as indicated by experiments demonstrating that the process is preserved in cells treated with the ATP synthase inhibitor oligomycin. Overall, we provide evidence that neuronal differentiation involves a shift from oxidative to fermentative metabolism, and that oxidative phosphorylation is not essential for this process.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/metabolismo , Glucosa/metabolismo , Neuronas/metabolismo , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Línea Celular Tumoral , Células Madre Embrionarias/citología , Inhibidores Enzimáticos/farmacología , Ratones , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Neuronas/citología , Oligomicinas/farmacología , Oxidación-Reducción/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacosRESUMEN
Increased plasma levels of free fatty acids (FFA) occur in states of insulin resistance such as obesity and type 2 diabetes mellitus. These high levels of plasma FFA are proposed to play an important role for the development of insulin resistance but the mechanisms involved are still unclear. This study investigated the effects of saturated and unsaturated FFA on insulin sensitivity in parallel with mitochondrial function. C2C12 myotubes were treated for 24 h with 0.1 mM of saturated (palmitic and stearic) and unsaturated (oleic, linoleic, eicosapentaenoic, and docosahexaenoic) FFA. After this period, basal and insulin-stimulated glucose metabolism and mitochondrial function were evaluated. Saturated palmitic and stearic acids decreased insulin-induced glycogen synthesis, glucose oxidation, and lactate production. Basal glucose oxidation was also reduced. Palmitic and stearic acids impaired mitochondrial function as demonstrated by decrease of both mitochondrial hyperpolarization and ATP generation. These FFA also decreased Akt activation by insulin. As opposed to saturated FFA, unsaturated FFA did not impair glucose metabolism and mitochondrial function. Primary cultures of rat skeletal muscle cells exhibited similar responses to saturated FFA as compared to C2C12 cells. These results show that in muscle cells saturated FFA-induced mitochondrial dysfunction associated with impaired insulin-induced glucose metabolism.
Asunto(s)
Ácidos Grasos/farmacología , Resistencia a la Insulina/fisiología , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Células Musculares/efectos de los fármacos , Células Musculares/patología , Músculo Esquelético/patología , Adenosina Trifosfato/biosíntesis , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Glucosa/metabolismo , Insulina/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/enzimología , Células Musculares/enzimología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/enzimología , Fibras Musculares Esqueléticas/patología , Oligomicinas/farmacología , Ácido Palmítico/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Ácidos Esteáricos/farmacologíaRESUMEN
As a protozoan parasite of hematophagous insects, Trypanosoma rangeli epimastigotes are exposed to reactive oxygen species during development in hosts. In this work, we investigated the role of H(2)O(2) as a modulator of the ecto-phosphatase activity present in living T. rangeli. We observed that H(2)O(2) inhibits ecto-phosphatase activities in the short and long epimastigote forms of T. rangeli. Ecto-phosphatase activity found in the short form was more sensitive than that found in the long form. Moreover, H(2)O(2) inhibited ecto-phosphatase activity of the short form in a dose-dependent manner and this inhibition was reversible after H(2)O(2) removal. This effect was not observed for T. rangeli ecto-ATPase, another ecto-enzyme present on the external surface of T. rangeli. Cysteine, beta-mercaptoethanol, and reduced glutathione were able to revert the enzyme inhibition promoted by H(2)O(2). Catalase and glutathione peroxidase stimulated this ecto-phosphatase activity, whereas superoxide dismutase was not able to modulate this activity. The ecto-phosphatase activity was also activated by FCCP and inhibited by oligomycin. It seems that H(2)O(2) plays a fundamental role in the regulation of cellular processes of these organisms. We showed, for the first time, that these parasites can produce H(2)O(2), and it is able to regulate ecto-phosphatase activity.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma/enzimología , Adenosina Trifosfatasas/metabolismo , Animales , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Catalasa/farmacología , Cisteína/farmacología , Activación Enzimática , Glutatión Peroxidasa/farmacología , Peróxido de Hidrógeno/farmacología , Mercaptoetanol/farmacología , Oligomicinas/farmacología , Superóxido Dismutasa/farmacologíaRESUMEN
As part of the innate immune response NK cells destroy infected, transformed, or otherwise stressed cells within hours of activation. In contrast, CD4(+) T lymphocytes require a sustained increase in their metabolism in order to cope with the biogenesis of cell components, in a process of proliferation and differentiation into effector cells. Recently, mitochondria have been implied in T lymphocyte immune synapse function but little is known on the role of mitochondria in the NK cell interaction with tumour cells. Here we analysed NK cells mitochondrial membrane potential (Deltapsi(m)) as an indicator of mitochondrial energy status and cellular homeostasis. Upon contact with K562 tumour cells, NK cells undergo Deltapsi(m) depolarization, indicating a rapid consumption of their metabolic energy. Furthermore, pharmacological inhibition of ATP synthesis down-regulates NK cell cytotoxic activity. Confocal- and electron-microscopy analyses showed re-organization of NK cells mitochondria towards the site of interaction with K562 tumour cell (NK cell immune synapse), perhaps as a way to compensate for local energy consumption. Interestingly, mitochondrial re-organization also takes place following NK stimulation with anti-NKGD2 antibodies but not with anti-KIR2DL1 antibodies, suggesting that activating rather than inhibiting cell signalling, triggered by NK cell receptors, is involved in NK cell mitochondria dynamics.
Asunto(s)
Sinapsis Inmunológicas/metabolismo , Células Asesinas Naturales/metabolismo , Mitocondrias/fisiología , Neoplasias/metabolismo , Anticuerpos Monoclonales , Citotoxicidad Inmunológica/efectos de los fármacos , Humanos , Inmunidad Innata , Células K562 , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/ultraestructura , Potencial de la Membrana Mitocondrial/inmunología , Microscopía Confocal , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Neoplasias/inmunología , Neoplasias/patología , Oligomicinas/farmacologíaRESUMEN
Alkylphosphocholines are a new class of anticancer agents. The mechanisms by which these drugs display their antitumor activities are not known. In this work, we show that erucylphosphohomocholine, a new antineoplastic compound, significantly decreased ATP synthesis in isolated rat liver mitochondria at a concentration of 50 microm or higher via permeabilization of the inner membrane. At a concentration of 25 microm, it induced a moderate swelling of mitochondria, a slight decrease of the inner membrane potential, and an increase in state 4 respiration without an essential influence on state 3 respiration or the outer membrane permeability to cytochrome c. We found that cyclosporin A did not prevent mitochondrial swelling induced by 25-100 microm erucylphosphohomocholine. Moreover, cyclosporin A induced a fast drop of the inner membrane potential in the presence of 25-50 microm erucylphosphohomocholine that seems to be due to a strong synergistic inhibition of the respiratory activity. The ratio of uncoupled to state 3 respiration rates increased from 1.3 +/- 0.1 with 25 microm erucylphosphohomocholine and from 1.5 +/- 0.1 with 1 microm cyclosporin A to 4.5 +/- 0.3 in the presence of both drugs. On the other hand, oligomycin or cyclosporin A protected certain cancer cell lines against erucylphosphohomocholine-induced apoptosis. This protection might be related to a prevention of cellular ATP hydrolysis by permeabilized mitochondria and to the inhibition of the classical permeability transition pore, respectively. Our findings provide new insight into the mechanisms by which these unusual alterations of mitochondria might be involved in anticancer activity of alkylphosphocholines.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ciclosporina/administración & dosificación , Sinergismo Farmacológico , Ácidos Erucicos/administración & dosificación , Mitocondrias/metabolismo , Consumo de Oxígeno , Fosforilcolina/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Ciclosporina/farmacología , Ácidos Erucicos/farmacología , Hidrólisis , Hígado/metabolismo , Masculino , Potenciales de la Membrana , Mitocondrias/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Oligomicinas/farmacología , Fosforilcolina/administración & dosificación , Fosforilcolina/química , Fosforilcolina/farmacología , RatasRESUMEN
An important antitumour effect of SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) has been shown. We now report the effects of this mesoionic compound on mitochondrial metabolism. SYD-1 (1.5 micromol mg(-1) protein) dose-dependently inhibited the respiratory rate by 65% and 40% in state 3 using sodium glutamate and succinate, respectively, as substrates. Phosphorylation efficiency was depressed by SYD-1, as evidenced by stimulation of the state 4 respiratory rate, which was more accentuated with glutamate ( approximately 180%) than with succinate ( approximately 40%), with 1.5 micromol mg(-1) protein of SYD-1. As a consequence of the effects on states 3 and 4, the RCC and ADP/O ratios were lowered by SYD-1 using both substrates, although this effect was stronger with glutamate. The formation of membrane electrical potential was inhibited by approximately 50% (1.5 micromol SYD-1mg(-1) protein). SYD-1 interfered with the permeability of the inner mitochondrial membrane, as demonstrated by assays of mitochondrial swelling in the presence of sodium acetate and valinomycin +K(+). SYD-1 (1.5 micromol mg(-1) protein) inhibited glutamate completely and succinate energized-mitochondrial swelling by 80% in preparations containing sodium acetate. The swelling of de-energized mitochondria induced by K(+) and valinomycin was inhibited by 20% at all concentrations of SYD-1. An analysis of the segments of the respiratory chain suggested that the SYD-1 inhibition site goes beyond the complex I and includes complexes III and IV. Glutamate dehydrogenase was inhibited by 20% with SYD-1 (1.5 micromol mg(-1) protein). The hydrolytic activity of complex F(1)F(o) ATPase in intact mitochondria was greatly increased ( approximately 450%) in the presence of SYD-1. Our results show that SYD-1 depresses the efficiency of electron transport and oxidative phosphorylation, suggesting that these effects may be involved in its antitumoural effect.
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Mitocondrias Hepáticas/efectos de los fármacos , Oxadiazoles/farmacología , Sidnonas/farmacología , Adenosina Trifosfatasas/metabolismo , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Respiración de la Célula/efectos de los fármacos , Iones/química , Masculino , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Dilatación Mitocondrial/efectos de los fármacos , Estructura Molecular , Oligomicinas/farmacología , Oxadiazoles/síntesis química , Ratas , Ratas Wistar , Sidnonas/químicaRESUMEN
OBJECTIVE: Mitochondrial calcium-activated K(+) (mitoK(Ca)) channels have been described as channels that are activated by Ca(2+), inner mitochondrial membrane depolarization and drugs such as NS-1619. NS-1619 is cardioprotective, leading to the assumption that this effect is related to the opening of mitoK(Ca) channels. Here, we show several weaknesses in this hypothesis. METHODS: Isolated mitochondria from rat hearts were tested for evidence of mitoK(Ca) activity by analyzing functional parameters in K(+)-rich and K(+)-free media. RESULTS: NS-1619 promoted mitochondrial depolarization both in K(+)-rich and K(+)-free media. Respiratory rate increments were also seen in the presence of NS-1619 for both media. In parallel, NS-1619 promoted respiratory inhibition, as evidenced by respiratory measurements in state 3. Mitochondrial volume measurements conducted using light scattering showed that NS-1619 led to swelling, in a manner unaltered by inhibitors of mitoK(Ca) channels, antagonists of adenosine triphosphate-sensitive potassium channels or inhibitors of the permeability transition. Swelling was also maintained when K(+) in the media was substituted with tetraethylammonium (TEA(+)), which is not transported by any known K(+) carrier. Electron microscopy experiments gave support to the idea that NS-1619-induced mitochondrial swelling took place in the absence of K(+). In addition to testing the pharmacological effects of NS-1619, we attempted, unsuccessfully, to promote mitoK(Ca) activity by altering Ca(2+) concentrations in the medium and inducing mitochondrial uncoupling. CONCLUSION: Our data indicate that NS-1619 promotes non-selective permeabilization of the inner mitochondrial membrane to ions, in addition to partial respiratory inhibition. Furthermore, we found no specific K(+) transport in isolated heart mitochondria compatible with mitoK(Ca) opening, whether by pharmacological or physiological stimuli. Our results indicate that NS-1619 has extensive mitochondrial effects unrelated to mitoK(Ca) and suggest that tissue protection mediated by NS-1619 may occur through mechanisms other than activation of these channels.
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Bencimidazoles/farmacología , Activación del Canal Iónico/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Canales de Potasio Calcio-Activados/efectos de los fármacos , Animales , Calcio/metabolismo , Calcio/farmacología , Medios de Cultivo , Ácido Glutámico/farmacología , Malatos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/ultraestructura , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , NADP/metabolismo , Oligomicinas/farmacología , Oxidación-Reducción , Consumo de Oxígeno , Fenazinas/farmacología , Potasio/metabolismo , Potasio/farmacología , Canales de Potasio Calcio-Activados/metabolismo , Ratas , Ratas Sprague-Dawley , Rotenona/farmacología , Sodio/metabolismo , Ácido Succínico/farmacología , Tetraetilamonio/metabolismo , Tetraetilamonio/farmacología , Técnicas de Cultivo de Tejidos , Desacopladores/farmacologíaRESUMEN
Mitochondrial Ca(2+) and its relation with the contraction induced by phenylephrine was investigated. In normal Ca(2+), carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone (FCCP) and oligomycin produced contraction similar to that promoted by phenylephrine. Phenylephrine-induced contraction was reduced by FCCP+oligomycin. In Ca(2+)-free, FCCP+oligomycin did not induce contraction. Response to FCCP+oligomycin was reduced upon Ca(2+) repletion and this response was lower than that to phenylephrine. Ca(2+) concentration was increased by FCCP+oligomycin. Since a profuse net of sarcoplasmic reticulum encloses mitochondria, a cross-talk between the two organelles may play an important role in the phenylephrine-induced contraction in presence of Ca(2+) encountered in both sarcoplasmic reticulum and extracellular medium of anococcygeus cells.
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Calcio/metabolismo , Comunicación Celular/fisiología , Mitocondrias Musculares/metabolismo , Músculo Liso/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Cafeína/farmacología , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Comunicación Celular/efectos de los fármacos , Combinación de Medicamentos , Fura-2/análogos & derivados , Fura-2/metabolismo , Soluciones Isotónicas , Masculino , Mitocondrias Musculares/efectos de los fármacos , Mitocondrias Musculares/ultraestructura , Contracción Muscular/efectos de los fármacos , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Oligomicinas/farmacología , Fenilefrina/farmacología , Potasio/farmacología , Ratas , Ratas Wistar , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/ultraestructura , Desacopladores/farmacologíaRESUMEN
In this work, we characterized an ecto-ATPase activity in intact mycelial forms of Fonsecaea pedrosoi, the primary causative agent of chromoblastomycosis. In the presence of 1 mM EDTA, fungal cells hydrolyzed adenosine-5'-triphosphate (ATP) at a rate of 84.6 +/- 11.3 nmol Pi h(-1) mg(-1) mycelial dry weight. The ecto-ATPase activity was increased at about five times (498.3 +/- 27.6 nmol Pi h(-1) mg(-1)) in the presence of 5 mM MgCl2, with values of Vmax and apparent Km for Mg-ATP(2-) corresponding to 541.9 +/- 48.6 nmol Pi h(-1) mg(-1) cellular dry weight and 1.9 +/- 0.2 mM, respectively. The Mg2+-stimulated ecto-ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A1(V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate, and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The surface of the Mg2+ -stimulated ATPase in F. pedrosoi was confirmed by assays in which 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid (DIDS), a membrane impermeant inhibitor, and suramin, an inhibitor of ecto-ATPase and antagonist of P2 purinoreceptors. Based on the differential expression of ecto-ATPases in the different morphological stages of F. pedrosoi, the putative role of this enzyme in fungal biology is discussed.