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1.
Nat Commun ; 12(1): 4835, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376679

RESUMO

F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ0 cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ0 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway.


Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Cálcio/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ionóforos de Próton/farmacologia
2.
Methods Mol Biol ; 2277: 391-403, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080164

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/farmacologia
3.
Nat Chem Biol ; 16(12): 1385-1393, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32778841

RESUMO

Mitochondrial membrane potential (ΔΨm) is a universal selective indicator of mitochondrial function and is known to play a central role in many human pathologies, such as diabetes mellitus, cancer and Alzheimer's and Parkinson's diseases. Here, we report the design, synthesis and several applications of mitochondria-activatable luciferin (MAL), a bioluminescent probe sensitive to ΔΨm, and partially to plasma membrane potential (ΔΨp), for non-invasive, longitudinal monitoring of ΔΨm in vitro and in vivo. We applied this new technology to evaluate the aging-related change of ΔΨm in mice and showed that nicotinamide riboside (NR) reverts aging-related mitochondrial depolarization, revealing another important aspect of the mechanism of action of this potent biomolecule. In addition, we demonstrated application of the MAL probe for studies of brown adipose tissue (BAT) activation and non-invasive in vivo assessment of ΔΨm in animal cancer models, opening exciting opportunities for understanding the underlying mechanisms and for discovery of effective treatments for many human pathologies.


Assuntos
Envelhecimento/genética , Diagnóstico por Imagem/métodos , Luciferina de Vaga-Lumes/química , Corantes Fluorescentes/química , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Potencial da Membrana Mitocondrial/genética , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/diagnóstico por imagem , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Dioxóis/farmacologia , Feminino , Luciferina de Vaga-Lumes/metabolismo , Corantes Fluorescentes/metabolismo , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Neoplasias Mamárias Experimentais/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Nigericina/farmacologia
4.
Biol Pharm Bull ; 43(8): 1210-1219, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32741941

RESUMO

Intracerebral hemorrhage (ICH) is a disease with high disability and mortality rates. Currently, the efficacy of therapies available for ICH is limited. Microglia-mediated neuroinflammation substantially exacerbates brain damage following ICH. Here, we investigated whether mitochondrial uncouplers conferred protection by suppressing neuroinflammation following ICH. To mimic ICH-induced neuroinflammation in vitro, we treated microglia with red blood cell (RBC) lysate. RBC lysate enhanced the expression of pro-inflammatory cytokines in microglia. A clinically used uncoupler, niclosamide (Nic), reduced the RBC lysate-induced expression of pro-inflammatory cytokines in microglia. Moreover, Nic ameliorated brain edema, decreased neuroinflammation, and improved neurological deficits in a well-established mouse model of ICH. Like niclosamide, the structurally unrelated uncoupler carbonyl cyanide p-triflouromethoxyphenylhydrazone (FCCP) reduced brain edema, decreased neuroinflammation, and improved neurological deficits following ICH. It has been reported that mitochondrial uncouplers activate AMP-activated protein kinase (AMPK). Mechanistically, Nic enhanced AMPK activation following ICH, and AMPK knockdown abolished the beneficial effects of Nic following ICH. In conclusion, mitochondrial uncouplers conferred protection by activating AMPK to inhibit microglial neuroinflammation following ICH.


Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Hemorragia Cerebral/tratamento farmacológico , Inflamação/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Niclosamida/farmacologia , Desacopladores/farmacologia , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cultivadas , Camundongos , Microglia/efeitos dos fármacos , Niclosamida/uso terapêutico
5.
Biochem Biophys Res Commun ; 530(1): 29-34, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32828301

RESUMO

Bicarbonate has been known to modulate activities of various mitochondrial enzymes such as ATPase and soluble adenylyl cyclase. Here, we found that the ability of conventional protonophoric uncouplers, such as 2,4-dinitrophenol (DNP), carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), but not that of the new popular uncoupler BAM15, to decrease mitochondrial membrane potential was significantly diminished in the presence of millimolar concentrations of bicarbonate. Thus, the depolarizing activity of DNP and FCCP in mitochondria could be sensitive to the local concentration of bicarbonate in cells and tissues. However, bicarbonate could not restore the ATP synthesis suppressed by DNP or CCCP in mitochondria. Bicarbonate neither altered the depolarizing action of DNP and FCCP on proteoliposomes with reconstituted cytochrome c oxidase, nor affected the protonophoric activity of DNP and FCCP in artificial lipid membranes as measured with pyranine-loaded liposomes, thereby showing that the bicarbonate-induced reversal of the depolarizing action of DNP and FCCP on mitochondria did not result from direct interaction of bicarbonate with the uncouplers.


Assuntos
Bicarbonatos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Desacopladores/farmacologia , 2,4-Dinitrofenol/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Ratos
6.
Sci Rep ; 10(1): 1569, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005875

RESUMO

Mitochondrial dysfunction is a hallmark in idiopathic Parkinson's disease (IPD). Here, we established screenable phenotypes of mitochondrial morphology and function in primary fibroblasts derived from patients with IPD. Upper arm punch skin biopsy was performed in 41 patients with mid-stage IPD and 21 age-matched healthy controls. At the single-cell level, the basal mitochondrial membrane potential (Ψm) was higher in patients with IPD than in controls. Similarly, under carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) stress, the remaining Ψm was increased in patients with IPD. Analysis of mitochondrial morphometric parameters revealed significantly decreased mitochondrial connectivity in patients with IPD, with 9 of 14 morphometric mitochondrial parameters differing from those in controls. Significant morphometric mitochondrial changes included the node degree, mean volume, skeleton size, perimeter, form factor, node count, erosion body count, endpoints, and mitochondria count (all P-values < 0.05). These functional data reveal that resistance to depolarization was increased by treatment with the protonophore FCCP in patients with IPD, whereas morphometric data revealed decreased mitochondrial connectivity and increased mitochondrial fragmentation.


Assuntos
Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/patologia , Doença de Parkinson/patologia , Idoso , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Estudos de Casos e Controles , Feminino , Fibroblastos/fisiologia , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Doença de Parkinson/fisiopatologia
7.
Autophagy ; 16(3): 562-574, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31234709

RESUMO

Selective elimination of mitochondria by autophagy is a critical strategy for a variety of physiological processes, including development, cell-fate determination and stress response. Although several mechanisms have been identified as responsible for selective degradation of mitochondria, such as the PINK1-PRKN/PARKIN- and receptor-dependent pathways, aspects of the mechanisms and particularly the principles underlying the selection process of mitochondria remain obscure. Here, we addressed a new selection strategy in which the selective elimination of mitochondria is dependent on organellar topology. We found that populations of mitochondria undergo different topological transformations under serum starvation, either swelling or forming donut shapes. Swollen mitochondria are associated with mitochondrial membrane potential dissipation and PRKN recruitment, which promote their selective elimination, while the donut topology maintains mitochondrial membrane potential and helps mitochondria resist autophagy. Mechanistic studies show that donuts resist autophagy even after depolarization through preventing recruitment of autophagosome receptors CALCOCO2/NDP52 and OPTN even after PRKN recruitment. Our results demonstrate topology-dependent, bifurcated mitochondrial recycling under starvation, that is swollen mitochondria undergo removal by autophagy, while donut mitochondria undergo fission and fusion cycles for reintegration. This study reveals a novel morphological selection for control of mitochondrial quality and quantity under starvation.


Assuntos
Mitocôndrias/metabolismo , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Mitocôndrias/ultraestrutura , Mitofagia/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
8.
Sensors (Basel) ; 19(14)2019 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-31330904

RESUMO

Electric cell-substrate impedance sensing (ECIS) is an emerging technique for sensitively monitoring morphological changes of adherent cells in tissue culture. In this study, human mesenchymal stem cells (hMSCs) were exposed to different concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) for 20 h and their subsequent concentration-dependent responses in micromotion and wound healing migration were measured by ECIS. FCCP disrupts ATP synthesis and results in a decrease in cell migration rates. To detect the change of cell micromotion in response to FCCP challenge, time-series resistances of cell-covered electrodes were monitored and the values of variance were calculated to verify the difference. While Seahorse XF-24 extracellular flux analyzer can detect the effect of FCCP at 3 µM concentration, the variance calculation of the time-series resistances measured at 4 kHz can detect the effect of FCCP at concentrations as low as 1 µM. For wound healing migration, the recovery resistance curves were fitted by sigmoid curve and the hill slope showed a concentration-dependent decline from 0.3 µM to 3 µM, indicating a decrease in cell migration rate. Moreover, dose dependent incline of the inflection points from 0.3 µM to 3 µM FCCP implied the increase of the half time for wound recovery migration. Together, our results demonstrate that partial uncoupling of mitochondrial oxidative phosphorylation reduces micromotion and wound healing migration of hMSCs. The ECIS method used in this study offers a simple and sensitive approach to investigate stem cell migration and its regulation by mitochondrial dynamics.


Assuntos
Técnicas de Cultura de Células , Impedância Elétrica , Células-Tronco Mesenquimais/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos
9.
Neuromolecular Med ; 21(4): 493-504, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31172441

RESUMO

Measuring mitochondrial respiration in brain tissue is very critical in understanding the physiology and pathology of the central nervous system. Particularly, measurement of respiration in isolated mitochondria provides the advantage over the whole cells or tissues as the changes in respiratory function are intrinsic to mitochondrial structures rather than the cellular signaling that regulates mitochondria. Moreover, a high-throughput technique for measuring mitochondrial respiration minimizes the experimental time and the sample-to-sample variation. Here, we provide a detailed protocol for measuring respiration in isolated brain non-synaptosomal mitochondria using Agilent Seahorse XFe24 Analyzer. We optimized the protocol for the amount of mitochondria and concentrations of ADP, oligomycin, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) for measuring respiratory parameters for complex I-mediated respiration. In addition, we measured complex II-mediated respiratory parameters. We observed that 10 µg of mitochondrial protein per well, ADP concentrations ranging between 2.5 and 10 mmol/L along with 5 µmol/L of oligomycin, and 5 µmol/L of FCCP are ideal for measuring the complex I-mediated respiration in isolated mouse brain mitochondria. Furthermore, we determined that 2.5 µg of mitochondrial protein per well is ideal for measuring complex II-mediated respiration. Notably, we provide a discussion of logical analysis of data and how the assay could be utilized to design mechanistic studies for experimental stroke. In conclusion, we provide detailed experimental design for measurement of various respiratory parameters in isolated brain mitochondria utilizing a novel high-throughput technique along with interpretation and analysis of data.


Assuntos
Encéfalo/metabolismo , Fluorometria/métodos , Ensaios de Triagem em Larga Escala/métodos , Microquímica/métodos , Mitocôndrias/metabolismo , Oximetria/métodos , Consumo de Oxigênio , Difosfato de Adenosina/farmacologia , Animais , Encéfalo/ultraestrutura , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Fluorometria/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microquímica/instrumentação , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/análise , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Oligomicinas/farmacologia , Fosforilação Oxidativa , Oximetria/instrumentação , Oxigênio/análise , Consumo de Oxigênio/efeitos dos fármacos , Prótons
10.
J Biol Chem ; 294(33): 12472-12482, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31248983

RESUMO

Type 2 taste receptors (TAS2R) are G protein-coupled receptors first described in the gustatory system, but have also been shown to have extraoral localizations, including airway smooth muscle (ASM) cells, in which TAS2R have been reported to induce relaxation. TAS2R46 is an unexplored subtype that responds to its highly specific agonist absinthin. Here, we first demonstrate that, unlike other bitter-taste receptor agonists, absinthin alone (1 µm) in ASM cells does not induce Ca2+ signals but reduces histamine-induced cytosolic Ca2+ increases. To investigate this mechanism, we introduced into ASM cells aequorin-based Ca2+ probes targeted to the cytosol, subplasma membrane domain, or the mitochondrial matrix. We show that absinthin reduces cytosolic histamine-induced Ca2+ rises and simultaneously increases Ca2+ influx into mitochondria. We found that this effect is inhibited by the potent human TAS2R46 (hTAS2R46) antagonist 3ß-hydroxydihydrocostunolide and is no longer evident in hTAS2R46-silenced ASM cells, indicating that it is hTAS2R46-dependent. Furthermore, these changes were sensitive to the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP); the mitochondrial calcium uniporter inhibitor KB-R7943 (carbamimidothioic acid); the cytoskeletal disrupter latrunculin; and an inhibitor of the exchange protein directly activated by cAMP (EPAC), ESI-09. Similarly, the ß2 agonist salbutamol also could induce Ca2+ shuttling from cytoplasm to mitochondria, suggesting that this new mechanism might be generalizable. Moreover, forskolin and an EPAC activator mimicked this effect in HeLa cells. Our findings support the hypothesis that plasma membrane receptors can positively regulate mitochondrial Ca2+ uptake, adding a further facet to the ability of cells to encode complex Ca2+ signals.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Sistema Respiratório/metabolismo , Sesquiterpenos de Guaiano/farmacologia , Cálcio/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Linhagem Celular , Retículo Endoplasmático/genética , Células HeLa , Humanos , Mitocôndrias/genética , Miócitos de Músculo Liso/citologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sistema Respiratório/citologia , Tioureia/análogos & derivados , Tioureia/farmacologia
11.
Anal Bioanal Chem ; 411(17): 3763-3768, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31093698

RESUMO

We describe a chip calorimetric technique that allows the investigation of biological material under anoxic conditions in a micro-scale and in real time. Due to the fast oxygen exchange through the sample flow channel wall, the oxygen concentration inside the samples could be switched between atmospheric oxygen partial pressure to an oxygen concentration of 0.5% within less than 2 h. Using this technique, anaerobic processes in the energy metabolism of Trypanosoma cruzi could be studied directly. The comparison of the calorimetric and respirometric response of T. cruzi cells to the treatment with the mitochondrial inhibitors oligomycin and antimycin A and the uncoupler FCCP revealed that the respiration-related heat rate is superimposed by strong anaerobic contributions. Calorimetric measurements under anoxic conditions and with glycolytic inhibitors showed that anaerobic metabolic processes contribute from 30 to 40% to the overall heat production rate. Similar basal and antimycin A heat rates with cells under anoxic conditions indicated that the glycolytic rates are independent of the oxygen concentration which confirms the absence of the "Pasteur effect" in Trypanosomes. Graphical abstract.


Assuntos
Calorimetria/métodos , Metabolismo Energético , Dispositivos Lab-On-A-Chip , Trypanosoma cruzi/metabolismo , Anaerobiose , Antimicina A/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Glicólise/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Oligomicinas/farmacologia , Oxigênio/metabolismo , Ionóforos de Próton/farmacologia
12.
Parasitol Res ; 118(4): 1127-1135, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30790039

RESUMO

Human infection due to eating fish parasitized by live Anisakis larvae in the third stage is considered an important health problem, and the application of treatments to ensure their mortality in the fish products is crucial to prevent the risk of infection. Mobility is used to assess viability, but mobile larvae may not always be infective and immobile larvae may be erroneously considered as non-viable. The objective was to establish whether the analysis of respiratory activity by means of the oxygen consumption rate (OCR) of Anisakis could be used to identify subtle differences between larvae that were still considered viable in terms of their mobility but had been subjected to thermal and/or chemical stress. The metabolic modulators FCCP [carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone] and sodium azide were used and the basal, maximum, spare and residual respiration rates calculated. Results showed that maximum respiratory capacity of larvae subjected to freezing significantly decreased immediately after thawing, but after some acclimatization, they recovered their capacity fully. However, when these larvae were stored at 4.6 °C, their mitochondria became dysfunctional faster than those of untreated larvae. OCR also showed that mitochondria of larvae were affected by incubation at 37 °C in NaCl or gastric juice. To conclude, OCR of Anisakis in the presence of metabolic modulators can help to identify subtle changes that occur in the larva. These measurements could be used to characterize larvae subjected to various stresses so that a broader picture of Anisakis pathogenic potential can be gained.


Assuntos
Anisakis/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Inibidores Enzimáticos/farmacologia , Larva/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio/fisiologia , Azida Sódica/farmacologia , Aclimatação/fisiologia , Animais , Anisaquíase/veterinária , Anisakis/embriologia , Doenças dos Peixes/parasitologia , Peixes/parasitologia , Humanos , Alimentos Marinhos/parasitologia , Cloreto de Sódio/farmacologia
13.
Acta Neuropathol Commun ; 6(1): 139, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30541620

RESUMO

Mesenchymal stem cells (MSCs) transfer healthy mitochondria to damaged acceptor cells via actin-based intercellular structures. In this study, we tested the hypothesis that MSCs transfer mitochondria to neural stem cells (NSCs) to protect NSCs against the neurotoxic effects of cisplatin treatment. Our results show that MSCs donate mitochondria to NSCs damaged in vitro by cisplatin. Transfer of healthy MSC-derived mitochondria decreases cisplatin-induced NSC death. Moreover, mitochondrial transfer from MSCs to NSCs reverses the cisplatin-induced decrease in mitochondrial membrane potential. Blocking the formation of actin-based intercellular structures inhibited the transfer of mitochondria to NSCs and abrogated the positive effects of MSCs on NSC survival. Conversely, overexpression of the mitochondrial motor protein Rho-GTPase 1 (Miro1) in MSCs increased mitochondrial transfer and further improved survival of cisplatin-treated NSCs.In vivo, MSC administration prevented the loss of DCX+ neural progenitor cells in the subventricular zone and hippocampal dentate gyrus which occurs as a result of cisplatin treatment. We propose mitochondrial transfer as one of the mechanisms via which MSCs exert their therapeutic regenerative effects after cisplatin treatment.


Assuntos
Cisplatino/farmacologia , Células-Tronco Mesenquimais/fisiologia , Mitocôndrias/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Neurotoxinas/farmacologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Toxina da Cólera/metabolismo , Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Neuropeptídeos/metabolismo , Oligomicinas/farmacologia , Tiazolidinas/farmacologia , Aglutininas do Germe de Trigo/metabolismo
14.
J Ovarian Res ; 11(1): 89, 2018 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-30326924

RESUMO

BACKGROUND: Cellular metabolic changes that accompany malignant transformation have been heralded as hallmark features of cancer. However, metabolic signatures between neoplasms can be unique, allowing for distinctions in malignancy, invasion and chemoresistance between cancer types and subtypes. Mitochondria are central metabolic mediators, as cellular bioenergetics veers from oxidative phosphorylation to glycolysis. Herein, we evaluate the role of mitochondria in maintenance of cellular metabolism, proliferation, and survival in the adult granulosa tumor cell line, KGN, as well as three epithelial ovarian cancer cell lines to determine distinctions in specific features. RESULTS: Notably, KGN cells were susceptible to TRAIL- and cisplatin-induced death following pretreatment with the metabolic inhibitor FCCP, but not oligomycin A. Collapse of mitochondrial membrane potential was found concomitant with cell death via apoptosis, independent from extrinsic canonical apoptotic routes. Rather, treatment with FCCP resulted in elevated cytochrome c release from mitochondria and decreased responsiveness to BIRC5. Following knockdown of BIRC5, mitochondrial membrane depolarization further sensitized KGN cells to induction of apoptosis via TRAIL. CONCLUSIONS: These results indicate an essential role, distinct from metabolism, for mitochondrial membrane potential in KGN cells to sense and respond to external mediators of apoptotic induction.


Assuntos
Tumor de Células da Granulosa/fisiopatologia , Potencial da Membrana Mitocondrial , Neoplasias Ovarianas/fisiopatologia , Survivina/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Morte Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Feminino , Humanos , Oligomicinas/farmacologia , Ionóforos de Próton/farmacologia , Survivina/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia
15.
Exp Cell Res ; 372(1): 61-72, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30236513

RESUMO

Thioredoxin 2 (Trx2), as a member of the thioredoxin system in mitochondria, is involved in controlling mitochondrial redox state. However, the role of Trx2 in cardiac biology is not fully understood. In the present study, the expression of Trx2 is silenced in quiescent neonatal rat ventricular cardiomyocytes (NRVCs) and mitochondrial respiratory function and cardiomyocyte hypertrophy are assessed. The results show that Trx2 depletion does not induce significant cytotoxicity in quiescent NRVCs. Remarkably, Trx2 depletion results in cardiomyocyte hypertrophy as determined by increased cell size and protein synthesis. Furthermore, Trx2 depletion inhibits AMPK activity and AMPK activator reversed cellular hypertrophy. Trx2 depletion enhances mitochondrial ROS generation without impact on cellular ROS level. Trx2 depletion has no effect on mitochondrial biogenesis. Specifically, Trx2 depletion increases mitochondrial respiration flux and total ATP concentration under quiescent conditions. To decipher the relationship between ROS generation, mitochondrial respiration flux, and AMPK signaling, mitochondrial metabolism and ROS was specifically inhibited, and the results show that AMPK inactivation and hypertrophic response in Trx2-silenced cells is reversed by respiration blockers but not ROS scavenger. In conclusion, these results show that beyond mitochondrial ROS scavenging, Trx2 controls mitochondrial respiratory function in quiescent cardiomyocytes and is implicated in cardiomyocyte hypertrophy via AMPK signaling.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Trifosfato de Adenosina/metabolismo , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Tamanho Celular , Regulação da Expressão Gênica , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Peptídeo Natriurético Encefálico/genética , Peptídeo Natriurético Encefálico/metabolismo , Oligomicinas/farmacologia , Oxirredução/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ribonucleotídeos/farmacologia , Rotenona/farmacologia , Transdução de Sinais , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/metabolismo
16.
Sci Rep ; 8(1): 12150, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30108299

RESUMO

Macrophage Migration Inhibitory Factor (MIF) is a multifunctional molecule highly secreted by human placenta mainly in the early phases of pregnancy. Studies in different cells show that MIF is a pro-survival factor by binding to its receptor CD74. By using the in vitro model of placental explants from first trimester pregnancy, we investigated the role of MIF in the survival of placental cells under induced stress conditions that promote apoptosis or mimic the hypoxia/re-oxygenation (H/R) injury that placenta could suffer in vivo. We demonstrated that recombinant MIF (rMIF) treatment was able to reduce caspase-3 activation when cultures were challenged with the apoptosis-inducer Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) while, in the cultures exposed to H/R, the treatment with rMIF did not show any effect. However, a significant increase in caspase-3 and caspase-8 activation was found when H/R-exposed cultures, were treated with anti-MIF or anti-CD74 antibody. We also observed that under H/R, a significant amount of endogenous MIF was released into the medium, which could account for the lack of effect of rMIF added to the cultures. Our results demonstrate for the first time that the MIF/CD74 axis contributes to maintain trophoblast homeostasis, by preventing abnormal apoptotic death.


Assuntos
Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Estresse Oxidativo/fisiologia , Primeiro Trimestre da Gravidez/fisiologia , Trofoblastos/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Apoptose/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Hipóxia Celular/fisiologia , Sobrevivência Celular/fisiologia , Feminino , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Gravidez , Proteínas Recombinantes/metabolismo , Técnicas de Cultura de Tecidos , Trofoblastos/efeitos dos fármacos
17.
Curr Protoc Cell Biol ; 79(1): e45, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29924486

RESUMO

This protocol describes how to apply appropriate pharmacological controls to induce mitochondrial fusion or fission in studies of mitochondria morphology for four different mammalian cell types, HepG2 human liver hepatocellular carcinoma cells, MCF7 human breast adenocarcinoma cells, HEK293 human embryonic kidney cells, and collagen sandwich culture of primary rat hepatocytes. The protocol provides methods of treating cells with these pharmacological controls, staining mitochondria with commercially available MitoTracker Green and TMRE dyes, and imaging the mitochondrial morphology in live cells using a confocal fluorescent microscope. It also describes the cell culture methods needed for this protocol. © 2018 by John Wiley & Sons, Inc.


Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Técnicas Citológicas/métodos , Mitocôndrias/metabolismo , Oligomicinas/farmacologia , Ribonucleotídeos/farmacologia , Aminoimidazol Carboxamida/farmacologia , Animais , Células HEK293 , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Indicadores e Reagentes , Células MCF-7 , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Ratos , Coloração e Rotulagem
18.
Cell Calcium ; 72: 1-17, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29748128

RESUMO

Interstitial cells of Cajal (ICC-MY) are pacemakers that generate and propagate electrical slow waves in gastrointestinal (GI) muscles. Slow waves appear to be generated by the release of Ca2+ from intracellular stores and activation of Ca2+-activated Cl- channels (Ano1). Conduction of slow waves to smooth muscle cells coordinates rhythmic contractions. Mitochondrial Ca2+ handling is currently thought to be critical for ICC pacemaking. Protonophores, inhibitors of the electron transport chain (FCCP, CCCP or antimycin) or mitochondrial Na+/Ca2+ exchange blockers inhibited slow waves in several GI muscles. Here we utilized Ca2+ imaging of ICC in small intestinal muscles in situ to determine the effects of mitochondrial drugs on Ca2+ transients in ICC. Muscles were obtained from mice expressing a genetically encoded Ca2+ indicator (GCaMP3) in ICC. FCCP, CCCP, antimycin, a uniporter blocker, Ru360, and a mitochondrial Na+/Ca2+ exchange inhibitor, CGP-37157 inhibited Ca2+ transients in ICC-MY. Effects were not due to depletion of ATP, as oligomycin did not affect Ca2+ transients. Patch-clamp experiments were performed to test the effects of the mitochondrial drugs on key pacemaker conductances, Ano1 and T-type Ca2+ (CaV3.2), in HEK293 cells. Antimycin blocked Ano1 and reduced CaV3.2 currents. CCCP blocked CaV3.2 current but did not affect Ano1 current. Ano1 and Cav3.2 currents were inhibited by CGP-37157. Inhibitory effects of mitochondrial drugs on slow waves and Ca2+ signalling in ICC can be explained by direct antagonism of key pacemaker conductances in ICC that generate and propagate slow waves. A direct obligatory role for mitochondria in pacemaker activity is therefore questionable.


Assuntos
Relógios Biológicos , Sinalização do Cálcio , Condutividade Elétrica , Células Intersticiais de Cajal/metabolismo , Intestino Delgado/citologia , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Anoctamina-1/metabolismo , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Canais de Cálcio Tipo T/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Clonazepam/análogos & derivados , Clonazepam/farmacologia , Células HEK293 , Humanos , Células Intersticiais de Cajal/citologia , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Músculos/citologia , Compostos de Rutênio/farmacologia , Tiazepinas/farmacologia
19.
Biochim Biophys Acta Bioenerg ; 1859(7): 491-500, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29625087

RESUMO

In changing light conditions, photosynthetic organisms develop different strategies to maintain a fine balance between light harvesting, photochemistry, and photoprotection. One of the most widespread photoprotective mechanisms consists in the dissipation of excess light energy in the form of heat in the photosystem II antenna, which participates to the Non Photochemical Quenching (NPQ) of chlorophyll fluorescence. It is tightly related to the reversible epoxidation of xanthophyll pigments, catalyzed by the two enzymes, the violaxanthin deepoxidase and the zeaxanthin epoxidase. In Phaeomonas sp. (Pinguiophyte, Stramenopiles), we show that the regulation of the heat dissipation process is different from that of the green lineage: the NPQ is strictly proportional to the amount of the xanthophyll pigment zeaxanthin and the xanthophyll cycle enzymes are differently regulated. The violaxanthin deepoxidase is already active in the dark, because of a low luminal pH, and the zeaxanthin epoxidase shows a maximal activity under moderate light conditions, being almost inactive in the dark and under high light. This light-dependency mirrors the one of NPQ: Phaeomonas sp. displays a large NPQ in the dark as well as under high light, which recovers under moderate light. Our results pinpoint zeaxanthin epoxidase activity as the prime regulator of NPQ in Phaeomonas sp. and therefore challenge the deepoxidase-regulated xanthophyll cycle dogma.


Assuntos
Diatomáceas/metabolismo , Xantofilas/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Diatomáceas/química , Luz , NADP/química , Oxirredutases/fisiologia , Fotoquímica , Xantofilas/química
20.
Biochim Biophys Acta Mol Cell Res ; 1865(4): 616-628, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29410069

RESUMO

Oxidative phosphorylation and glycolysis are important features, by which cells could bypass oxidative stress. The level of oxidative stress, and the ability of cells to promote oxidative phosphorylation or glycolysis, significantly determined proliferation or cell demise. In the present work, we have employed selective mitochondrial probe MitoTracker™ Orange CMTM/Ros (MTO) to estimate the level of oxidative stress in cancer cells at different stressed conditions. MTO is partially sensitive to decrease of mitochondrial membrane potential and to reactive oxygen species (ROS) generated in mitochondria. We have demonstrated, that fluorescence lifetime of MTO is much more sensitive to oxidative stress than intensity-based approaches. This method was validated in different cancer cell lines. Our approach revealed, at relatively low ROS levels, that Gö 6976, a protein kinase C (PKC) α inhibitor, and rottlerin, an indirect PKCδ inhibitor, increased mitochondrial ROS level in glioma cell. Their involvement in oxidative phosphorylation and apoptosis was investigated with oxygen consumption rate estimation, western blot and flow-cytometric analysis. Our study brings new insight to identify feeble differences in ROS production in living cells.


Assuntos
Glioma/patologia , Mitocôndrias/metabolismo , Imagem Molecular/métodos , Estresse Oxidativo , Acetofenonas/farmacologia , Antimicina A/farmacologia , Apoptose/efeitos dos fármacos , Benzopiranos/farmacologia , Carbazóis/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Linhagem Celular Tumoral , Citometria de Fluxo , Glioma/metabolismo , Glutationa/metabolismo , Humanos , Cinética , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Oligomicinas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Rotenona/farmacologia , Superóxidos/metabolismo , Fatores de Tempo
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