RESUMO
Mutations in the mitochondrial genome (mtDNA) are ubiquitous in humans and can lead to a broad spectrum of disorders. However, due to the presence of multiple mtDNA molecules in the cell, co-existence of mutant and wild-type mtDNAs (termed heteroplasmy) can mask disease phenotype unless a threshold of mutant molecules is reached. Importantly, the mutant mtDNA level can change across lifespan as mtDNA segregates in an allele- and cell-specific fashion, potentially leading to disease. Segregation of mtDNA is mainly evident in hepatic cells, resulting in an age-dependent increase of mtDNA variants, including non-synonymous potentially deleterious mutations. Here we modeled mtDNA segregation using a well-established heteroplasmic mouse line with mtDNA of NZB/BINJ and C57BL/6N origin on a C57BL/6N nuclear background. This mouse line showed a pronounced age-dependent NZB mtDNA accumulation in the liver, thus leading to enhanced respiration capacity per mtDNA molecule. Remarkably, liver-specific atg7 (autophagy related 7) knockout abolished NZB mtDNA accumulat ion, resulting in close-to-neutral mtDNA segregation through development into adulthood. prkn (parkin RBR E3 ubiquitin protein ligase) knockout also partially prevented NZB mtDNA accumulation in the liver, but to a lesser extent. Hence, we propose that age-related liver mtDNA segregation is a consequence of macroautophagic clearance of the less-fit mtDNA. Considering that NZB/BINJ and C57BL/6N mtDNAs have a level of divergence comparable to that between human Eurasian and African mtDNAs, these findings have potential implications for humans, including the safe use of mitochondrial replacement therapy.Abbreviations: Apob: apolipoprotein B; Atg1: autophagy-related 1; Atg7: autophagy related 7; Atp5a1: ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1; BL6: C57BL/6N mouse strain; BNIP3: BCL2/adenovirus E1B interacting protein 3; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mt-Atp8: mitochondrially encoded ATP synthase 8; MT-CO1: mitochondrially encoded cytochrome c oxidase I; MT-CO2: mitochondrially encoded cytochrome c oxidase II; mt-Co3: mitochondrially encoded cytochrome c oxidase III; mt-Cytb: mitochondrially encoded cytochrome b; mtDNA: mitochondrial DNA; MUL1: mitochondrial ubiquitin ligase activator of NFKB 1; nDNA: nuclear DNA; Ndufa9: NADH:ubiquinone oxireductase subunit A9; NDUFB8: NADH:ubiquinone oxireductase subunit B8; Nnt: nicotinamide nucleotide transhydrogenase; NZB: NZB/BINJ mouse strain; OXPHOS: oxidative phosphorylation; PINK1: PTEN induced putative kinase 1; Polg2: polymerase (DNA directed), gamma 2, accessory subunit; Ppara: peroxisome proliferator activated receptor alpha; Ppia: peptidylprolyl isomerase A; Prkn: parkin RBR E3 ubiquitin protein ligase; P10: post-natal day 10; P21: post-natal day 21; P100: post-natal day 100; qPCR: quantitative polymerase chain reaction; Rpl19: ribosomal protein L19; Rps18: ribosomal protein S18; SD: standard deviation; SEM: standard error of the mean; SDHB: succinate dehydrogenase complex, subunit B, iron sulfur (Ip); SQSTM1: sequestosome 1; Ssbp1: single-stranded DNA binding protein 1; TFAM: transcription factor A, mitochondrial; Tfb1m: transcription factor B1, mitochondrial; Tfb2m: transcription factor B2, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; UQCRC2: ubiquinol cytochrome c reductase core protein 2; WT: wild-type.
Assuntos
Mitofagia , NADP Trans-Hidrogenases , Trifosfato de Adenosina , Adulto , Animais , Apolipoproteínas/metabolismo , Apolipoproteínas B/metabolismo , Autofagia/genética , Dióxido de Carbono/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona , Citocromos b/metabolismo , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/metabolismo , Complexo III da Cadeia de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Ferro/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais , NAD/metabolismo , NADP Trans-Hidrogenases/metabolismo , PPAR alfa/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Ribossômicas/metabolismo , Proteína Sequestossoma-1/metabolismo , Succinato Desidrogenase/metabolismo , Enxofre/metabolismo , Fatores de Transcrição/metabolismo , Ubiquinona , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismoRESUMO
Trypanosoma cruzi is the causative agent of Chagas disease and has a single mitochondrion, an organelle responsible for ATP production and the main site for the formation of reactive oxygen species (ROS). T. cruzi is an obligate intracellular parasite with a complex life cycle that alternates between vertebrate and invertebrate hosts, therefore the development of survival strategies and morphogenetic adaptations to deal with the various environments is mandatory. Over the years our group has been studying the vector-parasite interactions using heme as a physiological oxidant molecule that triggered epimastigote proliferation however, the source of ROS induced by heme remained unknown. In the present study we demonstrate the involvement of heme in the parasite mitochondrial metabolism, decreasing oxygen consumption leading to increased mitochondrial ROS and membrane potential. First, we incubated epimastigotes with carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation, which led to decreased ROS formation and parasite proliferation, even in the presence of heme, correlating mitochondrial ROS and T. cruzi survival. This hypothesis was confirmed after the mitochondria-targeted antioxidant ((2-(2,2,6,6 Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO) decreased both heme-induced ROS and epimastigote proliferation. Furthermore, heme increased the percentage of tetramethylrhodamine methyl ester (TMRM) positive parasites tremendously-indicating the hyperpolarization and increase of potential of the mitochondrial membrane (ΔΨm). Assessing the mitochondrial functional metabolism, we observed that in comparison to untreated parasites, heme-treated epimastigotes decreased their oxygen consumption, and increased the complex II-III activity. These changes allowed the electron flow into the electron transport system, even though the complex IV (cytochrome c oxidase) activity decreased significantly, showing that heme-induced mitochondrial ROS appears to be a consequence of the enhanced mitochondrial physiological modulation. Finally, the parasites that were submitted to high concentrations of heme presented no alterations in the ultrastructure. Consequently, our results suggest that heme released by the insect vector after the blood meal, modify epimastigote mitochondrial physiology to increase ROS as a metabolic mechanism to maintain epimastigote survival and proliferation.
Assuntos
Doença de Chagas/imunologia , Heme/metabolismo , Mitocôndrias/metabolismo , Trypanosoma cruzi/fisiologia , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/metabolismo , Processos de Crescimento Celular , Células Cultivadas , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético , Humanos , Estágios do Ciclo de Vida , Potencial da Membrana Mitocondrial , Compostos Organofosforados/metabolismo , Consumo de Oxigênio , Piperidinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rodaminas/metabolismoRESUMO
BACKGROUND: Proliferation of Leishmania infantum depends on exogenous inorganic phosphate (P(i)) but little is known about energy metabolism and transport of P(i) across the plasma membrane in Leishmania sp. METHODS: We investigated the kinetics of 32P(i) transport, the influence of H+ and K+ ionophores and inhibitors, and expression of the genes for the Na+:P(i) and H+:P(i) cotransporters. RESULTS: The proton ionophore FCCP, bafilomycin A1 (vacuolar ATPase inhibitor), nigericin (K+ ionophore) and SCH28080 (an inhibitor of H+, K(+)-ATPase) all inhibited the transport of P(i). This transport showed Michaelis-Menten kinetics with K0.5 and V(max) values of 0.016 +/- 0.002 mM and 564.9 +/- 18.06 pmol x h(-1) x 10(-7) cells, respectively. These values classify the P(i) transporter of L. infantum among the high-affinity transporters, a group that includes Pho84 of Saccharomyces cerevisiae. Two sequences were identified in the L. infantum genome that code for phosphate transporters. However, transcription of the PHO84 transporter was 10-fold higher than the PHO89 transporter in this parasite. Accordingly, P(i) transport and LiPho84 gene expression were modulated by environmental P(i) variations. CONCLUSIONS: These findings confirm the presence of a P(i) transporter in L. infantum, similar to PHO84 in S. cerevisiae, that contributes to the acquisition of inorganic phosphate and could be involved in growth and survival of the promastigote forms of L. infantum. GENERAL SIGNIFICANCE: This work provides the first description of a PHO84-like P(i) transporter in a Trypanosomatide parasite of the genus Leishmania, responsible for many infections worldwide.
Assuntos
Leishmania infantum/enzimologia , Fosfatos/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Meios de Cultura , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Cinética , Leishmania infantum/genética , Macrolídeos/farmacologia , Dados de Sequência Molecular , Nigericina/farmacologia , Fosfatos/farmacologia , Radioisótopos de Fósforo , Filogenia , Ionóforos de Próton/farmacologia , Simportadores de Próton-Fosfato/antagonistas & inibidores , Simportadores de Próton-Fosfato/genética , Simportadores de Próton-Fosfato/metabolismo , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato/antagonistas & inibidores , Proteínas Cotransportadoras de Sódio-Fosfato/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Aging leads to progressive deterioration of physiological function and diminished responses to environmental stress. Organic and functional alterations are frequently observed in elderly subjects. Although chronic sleep loss is observed during senescence, little is known about the impact of insufficient sleep on cellular function in aging neurons. Disruption of neuronal calcium (Ca²âº) signaling is related to impaired neuronal function and cell death. It has been hypothesized that sleep deprivation may compromise neuronal stability and induce cell death in young neurons; however, it is necessary to evaluate the impact of aging on this process. Therefore, the aim of this study was to evaluate the effects of chronic sleep restriction (CSR) on Ca²âº signaling and cell death in the hippocampus of young and aged animals. We found that glutamate and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) induced a greater elevation in cytosolic Ca²âº ([Ca²âº](c)) in hippocampal slices from aged rats subjected to CSR compared to age-matched controls. Interestingly, aged-matched controls showed a reduced Ca²âº response to glutamate and FCCP, relative to both CSR and control young animals. Apoptotic nuclei were observed in aged rats from both treatment groups; however, the profile of apoptotic nuclei in aged CSR rats was highly variable. Bax and Bcl-2 protein expression did not change with aging in the CSR groups. Our study indicates that aging promotes changes in Ca²âº signaling, which may also be affected by CSR. These age-dependent changes in Ca²âº signaling may increase cellular vulnerability during CSR and contribute to Ca²âº signaling dysregulation, which may ultimately induce cell death.
Assuntos
Envelhecimento/fisiologia , Apoptose/fisiologia , Sinalização do Cálcio/fisiologia , Hipocampo/fisiopatologia , Privação do Sono/fisiopatologia , Envelhecimento/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Ácido Glutâmico/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Ratos , Ratos Wistar , Privação do Sono/metabolismo , Proteína X Associada a bcl-2/biossínteseRESUMO
Neuronal activity is accompanied by a rapid increase in interstitial lactate, which is hypothesized to serve as a fuel for neurons and a signal for local vasodilation. Using FRET microscopy, we report here that the rate of glycolysis in cultured mice astrocytes can be acutely modulated by physiological changes in extracellular lactate. Glycolytic inhibition by lactate was not accompanied by detectable variations in intracellular pH or intracellular ATP and was not dependent of mitochondrial function. Pyruvate was also inhibitory, suggesting that the effect of lactate is not mediated by the NADH/NAD(+) ratio. We propose that lactate serves as a fast negative feedback signal limiting its own production by astrocytes and therefore the amplitude of the lactate surge. The inhibition of glucose usage by lactate was much stronger in resting astrocytes than in K(+)-stimulated astrocytes, which suggests that lactate may also help diverting glucose from resting to active zones.
Assuntos
Astrócitos/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Ácido Láctico/farmacologia , Trifosfato de Adenosina/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Citocalasina B/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Concentração de Íons de Hidrogênio , Ácido Iodoacético/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Potássio/farmacologia , Ionóforos de Próton/farmacologia , Rotenona/farmacologiaRESUMO
Cytochrome c is known to play central role in apoptosis. Here, it is shown that ferricytochrome c, but not ferrocytochrome c is able to directly induce the aggregation of rat liver mitochondria, similar to the effect caused by magnesium ions at high concentrations. The aggregation was revealed by a decrease in light dispersion of mitochondrial suspension and it was confirmed by the optical microscopy. In the medium containing NADH and cytochrome c, mitochondrial aggregation was initiated only after exhaustion of NADH leading to oxidation of cytochrome c. The aggregation induced by 30 µM ferricytochrome c, but not by 5 mM MgCl(2), was completely inhibited by 30-100 µM ferricyanide, thus indicating that ferricyanide-cytochrome c specific interaction prevents mitochondrial aggregation. After completion of the aggregation caused by ferricytochrome c, this effect cannot be readily reversed by subsequent reduction of cytochrome c. The aggregation induced by ferricytochrome c and/or magnesium ions explains masking of the external NADH-oxidase activity of mitochondria in vitro reported in the literature. This new cytochrome c redox state-dependent phenomenon might also be involved in more complex mechanisms controlling aggregation (clustering) of mitochondria in vivo under the influence of pro-apoptotic factors and requires further study.
Assuntos
Citocromos c/farmacologia , Mitocôndrias Hepáticas/fisiologia , Animais , Antimicina A/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Citocromos c/química , Citocromos c/fisiologia , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Magnésio/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , NAD/farmacologia , NAD/fisiologia , Oxirredução , Ratos , Ratos Wistar , Espectrofotometria , Desacopladores/farmacologiaRESUMO
AIMS: Mitochondria are important modulators of Ca2+ homeostasis. However, it is not clear if they modulate and participate in smooth muscle signaling and contraction. The aim of the present work was to investigate the role of mitochondria in Ca2+ transients and contraction induced by metabotropic muscarinic receptor activation in rat gastric smooth muscle. MAIN METHODS: Carbachol (CCh)-induced contraction was investigated in the absence or presence of increasing concentration of mitochondrial protonophore, carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone (FCCP), in gastric fundus strips. Ca2+ and mitochondrial membrane potential (ΔΨm) measurements were performed in primarily cultured gastric smooth muscle cells loaded with FURA-2 or TMRE dyes. KEY FINDINGS: Results show that CCh (1 µM)-induced contraction was inhibited by FCCP in a concentration-dependent manner. In cultured smooth muscle cells CCh (1 µM) caused a cytosolic Ca2+ rise. Preincubation with FCCP strongly inhibited CCh-evoked Ca2+ transients indicating that mitochondria shape intracellular Ca2+ signals. CCh induced elevations of ∆Ψm in 60% of the individual mitochondrion analyzed. SIGNIFICANCE: Taken together our results indicate that CCh induces release of Ca2+ from intracellular stores, which may be modulated by mitochondria. Thus, mitochondria participate of the intracellular Ca2+ homeostasis in muscarinic contraction in gastric fundus smooth muscle.
Assuntos
Cálcio/metabolismo , Carbacol/farmacologia , Mucosa Gástrica/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Liso/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cultivadas , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Mitocôndrias Musculares/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Ratos , Ratos Wistar , Receptores Muscarínicos/efeitos dos fármacos , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Estômago/efeitos dos fármacos , Desacopladores/farmacologiaRESUMO
Huntington's disease is a neurodegenerative disorder caused by an expansion of CAGs repeats and characterized by alterations in mitochondrial functions. Although changes in Ca(2+) handling have been suggested, the mechanisms involved are not completely understood. The aim of this study was to investigate the possible alterations in Ca(2+) handling capacity and the relationship with mitochondrial dysfunction evaluated by NAD(P)H fluorescence, reactive oxygen species levels, mitochondrial membrane potential (DeltaPsi(m)) measurements and respiration in whole brain slices from R6/1 mice of different ages, evaluated in situ by real-time real-space microscopy. We show that the cortex and striatum of the 9-month-old R6/1 transgenic mice present a significant sustained increase in cytosolic Ca(2+) induced by glutamate (Glu). This difference in Glu response was partially reduced in R6/1 when in the absence of extracellular Ca(2+), indicating that N-methyl-D-aspartate receptors participation in this response is more important in transgenic mice. In addition, Glu also lead to a decrease in NAD(P)H fluorescence, a loss in DeltaPsi(m) and a further increase in respiration, which may have evoked a decrease in mitochondrial Ca(2+) Ca(2+)(m) uptake capacity. Taken together, these results show that alterations in Ca(2+) homeostasis in transgenic mice are associated with a decrease in Ca(2+)(m) uptake mechanism with a diminished Ca(2+) handling ability that ultimately causes dysfunctions and worsening of the neurodegenerative and the disease processes.
Assuntos
Encéfalo/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Mitocôndrias/metabolismo , Animais , Encéfalo/anatomia & histologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/metabolismo , Inibidores Enzimáticos/metabolismo , Homeostase , Humanos , Doença de Huntington/fisiopatologia , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Transgênicos , NADP/metabolismo , Consumo de Oxigênio/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Tapsigargina/metabolismo , Desacopladores/metabolismoRESUMO
Pronounced differences in the kinetics of single-vesicle catecholamine release from adrenal chromaffin cells stimulated with acetylcholine or high potassium (K(+)) have been recently found between normotensive Wistar rats (NWRs) and spontaneously hypertensive rats (SHRs). Such differences could be explained on the basis of distinct mechanisms of calcium (Ca(2+)) handling by chromaffin cells of NWRs and SHRs. We have explored here this hypothesis in adrenal medullary slices loaded with calcium fluorescent probes to measure the changes in Ca(2+) concentration in the cytosol ([Ca(2+)](c)), endoplasmic reticulum ([Ca(2+)](er)), and mitochondria ([Ca(2+)](m)). We found the following differences on calcium handling in SHRs, as compared with NWR: (i) higher basal [Ca(2+)](c) and basal [Ca(2+)](m); (ii) greater [Ca(2+)](c) elevations elicited by acetylcholine and K(+), with faster activation but slower inactivation; (iii) greater [Ca(2+)](c) elevations elicited by CRT (a mixture of caffeine, ryanodine, and thapsigargin) and by the mitochondrial protonophore FCCP (carbonylcyanide p-(trifluoromethoxy) phenylhydrazone). The higher basal [Ca(2+)](c) and [Ca(2+)](m) suggest an enhanced mitochondrial Ca(2+) uptake, and the greater [Ca(2+)](c) elevations produced by FCCP indicates a higher mitochondrial Ca(2+) release into the cytosol. This alteration of intracellular Ca(2+) movements could explain the greater quantal catecholamine release responses seen in SHRs, as compared with NWRs in previous studies. Furthermore, enhanced mitochondrial Ca(2+) cycling may be the basis for the dysfunction of mitochondrial bioenergetics, reported to be present in hypertensive states.
Assuntos
Medula Suprarrenal/citologia , Medula Suprarrenal/patologia , Cálcio/metabolismo , Citosol/metabolismo , Hipertensão/metabolismo , Hipertensão/patologia , Mitocôndrias/metabolismo , Acetilcolina/farmacologia , Medula Suprarrenal/efeitos dos fármacos , Medula Suprarrenal/metabolismo , Animais , Cafeína/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cromafins/citologia , Células Cromafins/efeitos dos fármacos , Células Cromafins/metabolismo , Células Cromafins/patologia , Citosol/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Fura-2/metabolismo , Técnicas In Vitro , Masculino , Movimento/efeitos dos fármacos , Potássio/farmacologia , Ratos , Ratos Endogâmicos SHR , Rianodina/farmacologia , Tapsigargina/farmacologiaRESUMO
Enzymatically dissociated mouse FDB muscle fibers, loaded with Fura-2 AM, were used to study the effect of mitochondrial uncoupling on the capacitative Ca(2+) entry, SOCE. Sarcoplasmic reticulum (SR) Ca(2+) stores were depleted by repetitive exposures to high K(+) or 4-chloro-m-Cresol (4-CmC) in the absence of extracellular Ca(2+). SR Ca(2+) store replenishment was substantially reduced using 5 microM cyclopiazonic acid (CPA). Readmission of external Ca(2+) (5 mM) increased basal [Ca(2+)](i) under two modalities. In mode 1 [Ca(2+)](i) initially increased at a rate of 0.8 +/- 0.1 nM/s and later at a rate of 12.3 +/- 2.6 nM/s, reaching a final value of 477.8 +/- 36.8 nM in 215.7 +/- 25.9 s. In mode 2, [Ca(2+)](i) increased at a rate of 0.8 +/- 0.1 nM/s to a value of 204.9 +/- 20.6 nM in 185.4 +/- 21.1 s. FCCP, 2 microM, reduced this Ca(2+) entry. In nine FCCP-poisoned fibers, the initial rate of Ca(2+) increase was 0.34 +/- 0.1 nM/s (mean +/- SEM), reaching a plateau of 149.2 +/- 14.1 nM in 217 +/- 19 s. The results may likely be explained by the hypothesis that SOCE is inhibited by mitochondrial uncouplers, pointing to a possible mitochondrial role in its activation. Using time-scan confocal microscopy and the dyes CaOr-5N AM or Rhod-2 AM to label mitochondrial Ca(2+), we show that during depletion [Ca(2+)](mito) initially increases and later diminishes. Finally, we show that the increase in basal [Ca(2+)](i), associated with SOCE activation, diminishes upon external Na(+) withdrawal. Na(+) entry through the SOCE pathway and activation of the reversal of Na(+)/Ca(2+) exchanger could explain this SOCE modulation by Na(+).
Assuntos
Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/toxicidade , Técnicas In Vitro , Indóis/farmacologia , Camundongos , Microscopia Confocal , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Modelos Biológicos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Trocador de Sódio e Cálcio/metabolismoRESUMO
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.
Assuntos
Peróxido de Hidrogênio/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma/enzimologia , Adenosina Trifosfatases/metabolismo , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Catalase/farmacologia , Cisteína/farmacologia , Ativação Enzimática , Glutationa Peroxidase/farmacologia , Peróxido de Hidrogênio/farmacologia , Mercaptoetanol/farmacologia , Oligomicinas/farmacologia , Superóxido Dismutase/farmacologiaRESUMO
Here, we present the first study on the effects of compounds that interfere with calcium (Ca(2+)) handling by the endoplasmic reticulum (ER) and mitochondria on amperometrically measured quantal catecholamine release from single adrenal chromaffin cells of control and spontaneously hypertensive rats (SHRs). Acetylcholine (ACh) or K(+) pulses triggered spike bursts of secretion by Ca(2+) entry through Ca(2+) channels. ER Ca(2+) release triggered by a mixture of caffeine, ryanodine, and thapsigargin (CRT) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (a mitochondrial protonophore) also caused bursts of secretory spikes. The spike bursts generated by ACh, K(+), CRT, and FCCP were 3 to 4 times longer in SHRs compared with control cells; furthermore, the individual spikes were faster and had 3-fold greater quantal size. In additional experiments, a 90-s treatment was made with CRT or FCCP to block Ca(2+) handling by the ER and mitochondria. In these conditions, the integrated spike burst responses elicited by ACh and K(+) were potentiated 2- to 3-fold in control and SHR cells. This suggests that variations in Ca(2+) entry and its subsequent redistribution into the ER and mitochondria are not responsible for the greater secretion seen in SHRs compared with control cells; rather, such differences seem to be due to greater quantal content of spike bursts and to greater quantal size of individual amperometric events.
Assuntos
Catecolaminas/metabolismo , Células Cromafins/metabolismo , Retículo Endoplasmático/metabolismo , Hipertensão/metabolismo , Mitocôndrias/metabolismo , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Separação Celular , Inibidores Enzimáticos/farmacologia , Potássio/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Rianodina/farmacologia , Tapsigargina/farmacologia , Desacopladores/farmacologiaRESUMO
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 report the use of the fluorescent dye CalciumOrange-5N (CaOr-5N) as a specific mitochondria Ca(2+) marker in enzymatically dissociated mouse FBD muscle fibers. Using laser scanning confocal microscopy and the dyes Mitotracker Green (MTG), di-8-ANEPPS and endoplasmic reticulum tracker green (ERTG), we determined the relative position of mitochondria, transverse tubules and sarcoplasmic reticulum in the sarcomere. Comparison with electron micrographies showed that mitochondria are mostly present at both sides of Z lines and near the triads located at the A-I band border. CaOr-5N fluorescence was mainly distributed in mitochondria, highly co-localised with MTG and basically excluded from the A band space. ERTG localised mostly between the two t-tubules present in each sarcomere. We studied the effect of the protonophore FCCP using CaOr-5N to measure mitochondrial Ca(2+) and JC-1 dye to measure mitochondria inner membrane potential (DeltaPsi(m)). After FCCP treatment, the CaOr-5N fluorescence diminished by about 33% in 80 s, while JC-1 fluorescence diminished by 36% in 200 s. Our results show the loss of Ca(2+) from mitochondria when DeltaPsi(m) is depolarised and demonstrate the usefulness of CaOr-5N to mark mitochondrial [Ca(2+)](m).
Assuntos
Cálcio/metabolismo , Corantes Fluorescentes , Microscopia Confocal , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Coloração e Rotulagem/métodos , Aldeídos , Animais , Benzimidazóis , Carbocianinas , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias Musculares/ultraestrutura , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/ultraestrutura , Compostos Orgânicos , Compostos de Piridínio , Sarcômeros/metabolismo , Retículo Sarcoplasmático/metabolismo , Fatores de Tempo , Desacopladores/farmacologiaRESUMO
We have studied the effects of mitochondria poisoning by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) on Ca(2+) signaling in enzymatically dissociated mouse flexor digitorum brevis (FDB) muscle fibers. We used Fura-2AM to measure resting [Ca(2+)](i) and MagFluo-4AM to measure Ca(2+) transients. Exposure to FCCP (2 microM, 2 min) caused a continuous increase in [Ca(2+)](i) at a rate of 0.60 nM/s and a drastic reduction of electrically elicited Ca(2+) transients without much effect on their decay phase. Half of the maximal effect occurred at [Ca(2+)](i) = 220 nM. This effect was partially reversible after long recuperation and was not diminished by Tiron, a reactive oxygen species (ROS) scavenger. FCCP had no effects on fiber excitability as shown by the generation of action potentials. 4CmC, an agonist of ryanodine receptors, induced a massive Ca(2+) release. FCCP diminished the rate but not the amount of Ca(2+) released, indicating that depletion of Ca(2+) stores did not cause the decrease in Ca(2+) transient amplitude. Ca(2+) transient amplitude could also be diminished, but to a lesser degree, by increases in [Ca(2+)](i) induced by repetitive stimulation of fibers treated with ciclopiazonic acid. This suggests an important role for Ca(2+) in the FCCP effect on transient amplitude.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/toxicidade , Mitocôndrias Musculares/efeitos dos fármacos , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Desacopladores/toxicidade , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/farmacologia , Potenciais de Ação , Animais , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Cresóis/farmacologia , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Sequestradores de Radicais Livres/farmacologia , Fura-2/análogos & derivados , Indóis/farmacologia , Cinética , Camundongos , Microscopia de Fluorescência/métodos , Mitocôndrias Musculares/metabolismo , Fibras Musculares de Contração Rápida/enzimologia , Fibras Musculares de Contração Rápida/metabolismo , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
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.
Assuntos
Cálcio/metabolismo , Comunicação Celular/fisiologia , Mitocôndrias Musculares/metabolismo , Músculo Liso/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Cafeína/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Comunicação Celular/efeitos dos fármacos , Combinação de Medicamentos , Fura-2/análogos & derivados , Fura-2/metabolismo , Soluções Isotônicas , Masculino , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/ultraestrutura , Contração Muscular/efeitos dos fármacos , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Oligomicinas/farmacologia , Fenilefrina/farmacologia , Potássio/farmacologia , Ratos , Ratos Wistar , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/ultraestrutura , Desacopladores/farmacologiaRESUMO
In this study, we investigated agents that increased intracellular calcium levels and their correlation with apoptotic cell death induction. We used rat astrocytes to investigate the increase in cytosolic Ca2+ (Ca(c)2+) and apoptosis induction by drugs that mobilize Ca2+ from different sources. We observed that thapsigargin (Thap), caffeine (Caff) and FCCP which caused similar increases in Ca(c)2+ levels (30-40%), also induced similar apoptotic rates (30-35%). On the other hand, antimycin (Anti), staurosporine (STS) and ethanol (Eth) promoted higher increases in Ca(c)2+ (55-65 %) and higher apoptotic rates (55-85%). Eth induced cell death in a concentration- and time-dependent manner. After treatment with Eth plus Caff for 6, 12 and 24 h, these effects were strongly potentiated. Results suggest that there might be a correlation between Ca(c)2+ increase and the rate of apoptosis. It is possible that Eth induces cell death by activation of more than one pathway and Ca2+ might be one of the elements involved. The present work indicates that Ca2+ can potentiate death by ethanol in rat astrocytes.
Assuntos
Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Cafeína/farmacologia , Cálcio/metabolismo , Depressores do Sistema Nervoso Central/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Animais , Animais Recém-Nascidos , Anexina A5/metabolismo , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Astrócitos/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cultivadas , Cloretos/farmacologia , Digitonina/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo/métodos , Fura-2 , Marcação In Situ das Extremidades Cortadas/métodos , Indicadores e Reagentes/farmacologia , Ionóforos/farmacologia , Compostos de Manganês/farmacologia , Propídio/metabolismo , Ratos , Estaurosporina/farmacologia , Tapsigargina/farmacologia , Fatores de TempoRESUMO
In this study, we investigated the effect of aging on intracellular Ca2+ stores, as sarcoendoplasmic reticulum (SR) and mitochondria, and the influence of these compartments on contraction of rat colon smooth muscle [Bitar, K.N., 2003. Aging and neural control of the GI tract V. Aging and gastrointestinal smooth muscle: from signal transduction to contractile proteins. Am. J. Physiol. Gastrointest. Liver. Physiol. 284(1), G1-G7; Marijic, J., Li, Q.X., Song, M., Nishimaru, K., Stefani, E., Toro, L., 2001. Decreased expression of voltage-and Ca2+-activated K+ channels in coronary smooth muscle during aging. Circ. Res. 88, 210-234; Rubio, C., Moreno, A., Briones, A. Ivorra, M.D., D'Ocon, P., Vila, E., 2002. Alterations by age of calcium handling in rat resistance arteries. J. Cardiovasc. Pharmacol. 40(6), 832-840]. Calcium stores and contraction were evaluated by simultaneous measurements of fluorescence and tension in smooth muscle strips loaded with fura-2. Results showed that activation of muscarinic receptors by methylcholine (MCh, 10 microM), induced a greater contraction in aged rats than in adult animals. The inhibition of Ca2+ ATPase by thapsigargin (TG, 1 microM) did not prevent the refilling of SR either in adult or aged rats. MCh, in the presence of TG, induced an increase in transient fluorescence, indicating a release of Ca2+ from TG-insensitive compartment. The mitochondrial uncoupler, FCCP (5 microM), caused a greater increase in intracellular Ca2+ and tension in aged rats, indicating that mitochondria may accumulate more Ca2+ during aging. The present results show that changes in intracellular Ca2+ stores, such as mitochondria and SR, affect contraction and may cause dysfunctions during aging that could culminate in severe alterations of Ca2+ homeostasis and cell damage.
Assuntos
Envelhecimento/metabolismo , Cálcio/metabolismo , Colo/metabolismo , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Envelhecimento/fisiologia , Animais , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/fisiologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Colina/análogos & derivados , Colina/farmacologia , Colo/efeitos dos fármacos , Colo/fisiologia , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Feminino , Mitocôndrias Musculares/metabolismo , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Ratos , Ratos Wistar , Retículo Sarcoplasmático/metabolismo , Tapsigargina/farmacologia , Técnicas de Cultura de Tecidos , Desacopladores/farmacologiaRESUMO
This study was designed to identify changes in endothelium-independent relaxation that could contribute to the depressed vascular reactivity and fall in blood pressure (BP) detected in rats after 5 weeks of streptozotocin (STZ)-induced (i.e. type 1) diabetes. Aortic rings were contracted by simultaneous activation of voltage-operated channels (KCl=80 mM) and alpha-adrenergic receptors (phenylephrine 1 microM) and then relaxed by simultaneous exposure to Ca2+-free PSS and 10 microM phentolamine. Additional relaxations were performed under conditions in which the plasma membrane Na-Ca exchanger (PMNaCa) or Ca-pump (PMCA), or the sarcoplasmic reticulum (SR) Ca-pump (SERCA) were blocked, to identify which mechanism(s) could modulate this process. The STZ-diabetic rats had a moderate but significant decrease of BP, and their aortic rings exhibited accelerated relaxation following a biexponential model, with a significantly decreased slow component. In control rats only the inhibition of the PMNaCa could slow down the fast component, while the slow component was insensitive to any blocking maneuver. In contrast, the diabetic animals' slow component was sensitive to the inhibition of both the PMNaCa and the SERCA. The SERCA-sensitive 45Ca2+ uptake by the SR was augmented in the aortas of STZ-treated animals. This hyperactivity of the SERCA, associated with augmented activity of the PMNaCa, at least partly induced by an increase of the plasma membrane Na+/K+-ATPase activity, could explain the decrease in BP and the accelerated aortic relaxation observed in the diabetic rats.
Assuntos
Pressão Sanguínea/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Músculo Liso Vascular/fisiopatologia , Vasodilatação/fisiologia , Animais , Aorta , Glicemia/metabolismo , Peso Corporal , Cálcio/fisiologia , Sinalização do Cálcio , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Modelos Animais de Doenças , Ácido Egtázico/farmacologia , Endotélio Vascular/fisiopatologia , Feminino , Músculo Liso Vascular/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Bisphenols are a class of compounds that exhibit a broad spectrum of antimicrobial activity. One of the most widely used member of this group is triclosan (TRN). TRN is a synthetic, non-ionic, broad-spectrum antimicrobial agent, which is incorporated into several products, including hand soaps and detergents and those of skin care and oral hygiene. The effects of TRN on mitochondrial respiratory parameters and the inner mitochondrial membrane potential (DeltaPsi) are described. That of TRN (up to 60 nmol mg(-1) protein) on isolated liver mitochondria decreased oxygen consumption of state 3 respiration, as well as DeltaPsi, but increased oxygen consumption of state 4 respiration, characteristic of an uncoupler effect. Analysis of segments of the respiratory chain suggested that the TRN inhibition site is located between complexes II and III. Mitochondrial swelling, energized or driven by the K+ diffusion potential using valinomycin, was also inhibited by TRN, the former being completely inhibited at concentrations greater than 10 nmol TRN mg(-1) protein, suggesting that it is also able to interfere with fluidity of the inner mitochondrial membrane. These results suggest that, besides its antibacterial effect, TRN can also impair the mitochondrial function of animal cells.