RESUMO
When mitochondria are exposed to high Ca2+ concentrations, especially when accompanied by oxidative stress and adenine nucleotide depletion, they undergo massive swelling and become uncoupled. This occurs as a result of the opening of a non-specific pore in the inner mitochondrial membrane, known as the MPTP (mitochondrial permeability transition pore). If the pore remains open, cells cannot maintain their ATP levels and this will lead to cell death by necrosis. This article briefly reviews what is known of the molecular mechanism of the MPTP and its role in causing the necrotic cell death of the heart and brain that occurs during reperfusion after a long period of ischaemia. Such reperfusion injury is a major problem during cardiac surgery and in the treatment of coronary thrombosis and stroke. Prevention of MPTP opening either directly, using agents such as cyclosporin A, or indirectly by reducing oxidative stress or Ca2+ overload, provides a protective strategy against reperfusion injury. Furthermore, mice in which a component of the MPTP, CyP-D (cyclophilin D), has been knocked out are protected against heart and brain ischaemia/reperfusion. When cells experience a less severe insult, the MPTP may open transiently. The resulting mitochondrial swelling may be sufficient to cause release of cytochrome c and activation of the apoptotic pathway rather than necrosis. However, the CyP-D-knockout mice develop normally and show no protection against a range of apoptotic stimuli, suggesting that the MPTP does not play a role in most forms of apoptosis.
Assuntos
Apoptose , Cálcio/metabolismo , Mitocôndrias/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Permeabilidade da Membrana Celular , Membranas Mitocondriais/metabolismoRESUMO
Tumour necrosis factor-alpha (TNF-alpha) may activate both cell survival and cell death pathways. In the murine fibrosarcoma cell line WEHI-164, physiological concentrations (1 ng/ml) of TNF-alpha induced wortmannin-sensitive cell ruffling characteristic of the phosphoinositide 3-kinase (PI3-kinase) activation associated with cell survival. Wortmannin also enhanced cell death induced by TNF-alpha in the presence of actinomycin D, confirming that TNF-alpha activates a transcription-independent survival pathway requiring PI3-kinase activity. Both TNF-alpha and insulin-like growth factor 1 (IGF-1) caused a 6-10-fold wortmannin-sensitive increase in protein kinase B (PKB) activity within 5 min. For IGF-1, this was associated with an increase in phosphorylation of both Thr(308) and Ser(473), whereas for TNF-alpha only phosphorylation of Ser(473) was increased, even in the presence of okadaic acid to inhibit protein phosphatases 1 and 2A. TNF-alpha did not decrease the phosphorylation of Thr(308) induced by IGF-1, implying that TNF-alpha neither inhibits phosphoinositide-dependent kinase 1 (PDK1) nor activates an opposing phosphatase. In WEHI cells overexpressing a form of PKB, IGF-1 increased phosphorylation of Ser(473) on PKB, but not its kinase activity, whereas TNF-alpha failed to induce Ser(473) phosphorylation or kinase activation of either overexpressed T308A or wild-type PKB (where T308A is the mutant bearing the substitution Thr(308)-->A). IGF-1 caused translocation of green-fluorescent-protein-tagged ADP-ribosylation factor nucleotide-binding site opener (ARNO) to the plasma membrane of WEHI cells, but this was not detected with TNF-alpha. We conclude that, at physiological concentrations, TNF-alpha activates endogenous PKB by stimulating PDK2 (increase in Ser(473) phosphorylation) in a PI3-kinase-dependent (wortmannin-sensitive) manner, without causing detectable stimulation of PDK1 (no increase in Thr(308) phosphorylation) or ARNO translocation. Possible explanations of these observations are discussed.
Assuntos
Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Serina/metabolismo , Treonina/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Linhagem Celular , Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Ácido Okadáico/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
The ischemic heart requires reperfusion using clinical interventions, such as coronary artery bypass graft surgery, in order to recover. Despite recent developments in myocardial protection techniques, reperfusion damage still occurs, and significant morbidity remains a problem. Therefore, the search continues for techniques that will limit myocardial damage and that will enhance recovery upon reperfusion. Mitochondria are known to be intimately involved in the processes that lead to cell death following reperfusion, in both necrotic and apoptotic forms of cell death, and so are potential targets for protective intervention. In this review, we consider several aspects of mitochondrial function that we believe to be possible targets for myocardial protection; namely, mitochondrial Ca(2+) transport, the permeability transition pore, and improved mitochondrial substrate supply. We discuss work by ourselves and others in these areas, and also consider the recently proposed role of mitochondrial ATP-dependent K(+) channels in mediating myocardial protection by ischemic preconditioning. Finally, we describe use of cardioplegic solutions in the clinical setting, and discuss how improved understanding of the aspects of mitochondrial function summarised above may lead to better protective strategies in the future.
Assuntos
Cálcio/metabolismo , Mitocôndrias Cardíacas/fisiologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Apoptose , Humanos , Mitocôndrias Cardíacas/metabolismo , Isquemia Miocárdica/fisiopatologia , Miocárdio/metabolismoRESUMO
Monocarboxylate transporter (MCT) 4 is the major monocarboxylate transporter isoform present in white skeletal muscle and is responsible for the efflux of lactic acid produced by glycolysis. Here we report the characterisation of MCT4 expressed in Xenopus oocytes. The protein was correctly targeted to the plasma membrane and rates of substrate transport were determined from the rate of intracellular acidification monitored with the pH-sensitive dye 2', 7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In order to validate the technique, the kinetics of monocarboxylate transport were measured in oocytes expressing MCT1. Km values determined for L-lactate, D-lactate and pyruvate of 4.4, > 60 and 2.1 mM, respectively, were similar to those determined previously in tumour cells. Comparison of the time course of [14C]lactate accumulation with the rate of intracellular acidification monitored with BCECF suggests that the latter reflects pH changes close to the plasma membrane associated with transport, whilst the former may include diffusion-limited movement of lactate into the bulk cytosol. Km values of MCT4 for these substrates were found to be 28, 519 and 153 mM, respectively, and for a range of other monocarboxylates values were at least an order of magnitude higher than for MCT1. Vmax values appeared to be similar for all substrates. K0.5 values of MCT4 (determined at 30 mM L-lactate) for inhibition by alpha-cyano-4-hydroxycinnamate (991 microM), phloretin (41 microM), 5-nitro-2-(3-phenylpropylamino)benzoate (240 microM), p-chloromercuribenzene sulphonate (21 microM) and 3-isobutyl-1-methylxanthine (970 microM, partial inhibition) were also substantially higher than for MCT1. No inhibition of MCT4 by 2 mM 4,4'-diisothiocyanostilbene-2,2'-disulphonate was observed. The properties of MCT4 are consistent with published data on giant sarcolemmal vesicles in which MCT4 is the dominant MCT isoform, and are appropriate for the proposed role of MCT4 in mediating the efflux from the cell of glycolytically derived lactic acid but not pyruvate.
Assuntos
Proteínas de Transporte/fisiologia , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos , Proteínas Musculares , Músculo Esquelético/enzimologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Células Cultivadas , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Cinética , Músculo Esquelético/metabolismo , Oócitos/metabolismo , Isoformas de Proteínas/fisiologia , Especificidade por Substrato , XenopusRESUMO
CD147 is a broadly expressed plasma membrane glycoprotein containing two immunoglobulin-like domains and a single charge-containing transmembrane domain. Here we use co-immunoprecipitation and chemical cross-linking to demonstrate that CD147 specifically interacts with MCT1 and MCT4, two members of the proton-linked monocarboxylate (lactate) transporter family that play a fundamental role in metabolism, but not with MCT2. Studies with a CD2-CD147 chimera implicate the transmembrane and cytoplasmic domains of CD147 in this interaction. In heart cells, CD147 and MCT1 co-localize, concentrating at the t-tubular and intercalated disk regions. In mammalian cell lines, expression is uniform but cross-linking with anti-CD147 antibodies caused MCT1, MCT4 and CD147, but not GLUT1 or MCT2, to redistribute together into 'caps'. In MCT-transfected cells, expressed protein accumulated in a perinuclear compartment, whereas co-transfection with CD147 enabled expression of active MCT1 or MCT4, but not MCT2, in the plasma membrane. We conclude that CD147 facilitates proper expression of MCT1 and MCT4 at the cell surface, where they remain tightly bound to each other. This association may also be important in determining their activity and location.
Assuntos
Antígenos CD , Antígenos de Neoplasias , Antígenos de Superfície/metabolismo , Proteínas Aviárias , Proteínas Sanguíneas , Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Musculares , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/isolamento & purificação , Basigina , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Membrana Celular/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Chaperonas Moleculares/metabolismo , Transportadores de Ácidos Monocarboxílicos , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Testes de Precipitina , Ligação Proteica , Ratos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Although metformin is widely used for the treatment of non-insulin-dependent diabetes, its mode of action remains unclear. Here we provide evidence that its primary site of action is through a direct inhibition of complex 1 of the respiratory chain. Metformin(50 microM) inhibited mitochondrial oxidation of glutamate+malate in hepatoma cells by 13 and 30% after 24 and 60 h exposure respectively, but succinate oxidation was unaffected. Metformin also caused time-dependent inhibition of complex 1 in isolated mitochondria, whereas in sub-mitochondrial particles inhibition was immediate but required very high metformin concentrations (K(0.5),79 mM). These data are compatible with the slow membrane-potential-driven accumulation of the positively charged drug within the mitochondrial matrix leading to inhibition of complex 1. Metformin inhibition of gluconeogenesis from L-lactate in isolated rat hepatocytes was also time- and concentration-dependent, and accompanied by changes in metabolite levels similar to those induced by other inhibitors of gluconeogenesis acting on complex 1. Freeze-clamped livers from metformin-treated rats exhibited similar changes in metabolite concentrations. We conclude that the drug's pharmacological effects are mediated, at least in part, through a time-dependent, self-limiting inhibition of the respiratory chain that restrains hepatic gluconeogenesis while increasing glucose utilization in peripheral tissues. Lactic acidosis, an occasional side effect, canal so be explained in this way.
Assuntos
Hipoglicemiantes/farmacologia , Metformina/farmacologia , Mitocôndrias/efeitos dos fármacos , NADH NADPH Oxirredutases/antagonistas & inibidores , Animais , Transporte de Elétrons/efeitos dos fármacos , Complexo I de Transporte de Elétrons , Gluconeogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Mitocôndrias/enzimologia , Ratos , Ratos Wistar , Transdução de Sinais , Partículas Submitocôndricas/enzimologia , Células Tumorais CultivadasRESUMO
Mitochondria play a central role in both apoptosis and necrosis through the opening of the mitochondrial permeability transition pore (MPTP). This is thought to be formed through a Ca(2+)-triggered conformational change of the adenine nucleotide translocase (ANT) bound to matrix cyclophilin-D and we have now demonstrated this directly by reconstitution of the pure components. Opening of the MPTP causes swelling and uncoupling of mitochondria which, unrestrained, leads to necrosis. In ischaemia/reperfusion injury of the heart we have shown MPTP opening directly. Recovery of hearts correlates with subsequent closure, and agents that prevent opening or enhance closure protect from injury. Transient MPTP opening may also be involved in apoptosis by initially causing swelling and rupture of the outer membrane to release cytochrome c (cyt c), which then activates the caspase cascade and sets apoptosis in motion. Subsequent MPTP closure allows ATP levels to be maintained, ensuring that cell death remains apoptotic rather than necrotic. Apoptosis in the hippocampus that occurs after a hypoglycaemic or ischaemic insult is triggered by this means. Other apoptotic stimuli such as cytokines or removal of growth factors also involve mitochondrial cyt c release, but here there is controversy over whether the MPTP is involved. In many cases cyt c release is seen without any mitochondrial depolarization, suggesting that the MPTP does not open. Recent data of our own and others have revealed a specific outer-membrane cyt c-release pathway involving porin that does not release other intermembrane proteins such as adenylate kinase. This is opened by pro-apoptotic members of the Bcl-2 family such as BAX and prevented by anti-apoptotic members such as Bcl-X(L). Our own data suggest that this pathway may interact directly with the ANT in the inner membrane at contact sites.
Assuntos
Apoptose , Mitocôndrias/fisiologia , Animais , Caspase 3 , Caspases/biossíntese , Dextranos/metabolismo , Ativação Enzimática , Humanos , Camundongos , Mitocôndrias Cardíacas/metabolismo , Modelos Biológicos , Necrose , Concentração Osmolar , Ratos , Fatores de Tempo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Percoll-purified rat liver mitochondria were shown to contain BAX dimer and rapidly (<2 min) release 5-10% of their cytochrome c when incubated in a standard KCl incubation medium under energized conditions. This release was not accompanied by release of adenylate kinase (AK), another intermembrane protein, and was not inhibited by Mg(2+), dATP, inhibitors of the permeability transition or ligands of the peripheral benzodiazepine receptor. However, release was greatly reduced by the presence of 5% (w/v) dextran (40 kDa), which caused a decrease in the light scattering (A(520)) of mitochondrial suspensions. Dextran also inhibited both mitochondrial oxidation of exogenous ferrocytochrome c in the presence of rotenone and antimycin, and respiratory-chain-driven reduction of exogenous ferricytochrome c. Hypo-osmotic medium or digitonin treatment of mitochondria caused a large additional release of both cytochrome c and AK that was not blocked by dextran. Polyaspartate, which stabilizes the low conductance state of the voltage-dependent anion channel (VDAC), increased cytochrome c release. VDAC and BAX are both found at the contact sites between the inner and outer membranes and dextran is known to stabilize these contact sites in isolated mitochondria. Thus our data suggest that regulation of a specific permeability pathway for cytochrome c may be mediated by changes in protein-protein interactions within contact sites. The adenine nucleotide translocase is known to bind to VDAC and thus provides an additional link between the specific cytochrome c release pathway and the permeability transition.
Assuntos
Grupo dos Citocromos c/metabolismo , Membranas Intracelulares/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Adenilato Quinase/metabolismo , Animais , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Ciclosporina/farmacologia , Dextranos/farmacologia , Dimerização , Membranas Intracelulares/efeitos dos fármacos , Cinética , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Peptídeos/farmacologia , Permeabilidade , Porinas/metabolismo , Cloreto de Potássio/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Wistar , Rotenona/farmacologia , Sacarose/farmacologia , Canais de Ânion Dependentes de Voltagem , Proteína X Associada a bcl-2RESUMO
This study evaluated macrophage expression of the stereospecific lactate transporter, MCT1, and the effects of lipopolysaccharide (LPS), tumor necrosis factor (TNFa), or nitric oxide (NO) on MCT1 mRNA and protein levels and lactate transporter activity. Peritoneal and J774.1 macrophages were treated with either saline, LPS (10 or 100 ng/mL), dexamethasone (100 nM), and dexamethasone + LPS. Cells were harvested at 4, 8, and 16 h after treatment and processed for total RNA and protein isolation. LPS treatment significantly increased macrophage MCT1 mRNA expression at 4 and 8 h compared with the saline-treated cells. Dexamethasone did not alter MCT1 mRNA levels. Treatment of J774.1 macrophages with TNFalpha (1 ng/mL) or nitric oxide (DETA-NO, 100 microM) also significantly increased MCT1 mRNA levels at 4 and 8 h after treatment. LPS and TNFalpha treatment significantly increased MCT1 protein levels at 8 and 16 h after treatment. Lactate transporter activity in J774.1 macrophages was measured by uptake of 14C-labeled lactate. LPS and TNFalpha treatment significantly augmented lactate uptake, 12 h after administration; however, nitric oxide treatment did not affect lactate uptake. Thus, our data demonstrated that stimulated peritoneal and J774.1 macrophages express the lactate transporter, MCT, and that LPS and TNFalpha regulate MCT1 mRNA and protein levels.
Assuntos
Proteínas de Transporte/genética , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/metabolismo , Macrófagos/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Ácidos Cumáricos/farmacologia , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Cinética , Lactatos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Transportadores de Ácidos Monocarboxílicos , Doadores de Óxido Nítrico/farmacologia , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacos , Triazenos/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: Diminishing oxidative stress may protect the heart against ischaemia-reperfusion injury by preventing opening of the mitochondrial permeability transition (MPT) pore. The general anaesthetic agent propofol, a free radical scavenger, has been investigated for its effect on the MPT and its cardioprotective action following global and cardioplegic ischaemic arrest. METHOD: Isolated perfused Wistar rat hearts were subjected to either warm global ischaemia (Langendorff) or cold St. Thomas' cardioplegia (working heart mode) in the presence or absence of propofol. MPT pore opening was determined using [3H]-2-deoxyglucose-6-phosphate ([3H]-DOG-6P) entrapment. The respiratory function of isolated mitochondria was also determined for evidence of oxidative stress. RESULTS: Propofol (2 micrograms/ml) significantly improved the functional recovery of Langendorff hearts on reperfusion (left ventricular developed pressure from 28.4 +/- 6.2 to 53.3 +/- 7.3 mmHg and left ventricular end diastolic pressure from 52.9 +/- 4.3 to 37.5 +/- 3.9 mmHg). Recovery was also improved in propofol (4 micrograms/ml) treated working hearts following cold cardioplegic arrest. External cardiac work on reperfusion improved from 0.42 +/- 0.05 to 0.60 +/- 0.03 J/s, representing 45-64% of baseline values, when compared to controls (P < 0.05). Propofol inhibited MPT pore opening during reperfusion, [3H]-DOG-6P entrapment being 16.7 vs. 22.5 ratio units in controls (P < 0.05). Mitochondria isolated from non-ischaemic, propofol-treated hearts exhibited increased respiratory chain activity and were less sensitive to calcium-induced MPT pore opening. CONCLUSION: Propofol confers significant protection against global normothermic ischaemia and during cold cardioplegic arrest. This effect is associated with less opening of mitochondrial MPT pores, probably as a result of diminished oxidative stress. Propofol may be a useful adjunct to cardioplegic solutions in heart surgery.
Assuntos
Sequestradores de Radicais Livres/uso terapêutico , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Propofol/uso terapêutico , Análise de Variância , Animais , Emulsões , Emulsões Gordurosas Intravenosas , Parada Cardíaca Induzida , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Perfusão , Fosfolipídeos , Ratos , Ratos Wistar , Óleo de SojaRESUMO
Monocarboxylates such as lactate and pyruvate play a central role in cellular metabolism and metabolic communication between tissues. Essential to these roles is their rapid transport across the plasma membrane, which is catalysed by a recently identified family of proton-linked monocarboxylate transporters (MCTs). Nine MCT-related sequences have so far been identified in mammals, each having a different tissue distribution, whereas six related proteins can be recognized in Caenorhabditis elegans and 4 in Saccharomyces cerevisiae. Direct demonstration of proton-linked lactate and pyruvate transport has been demonstrated for mammalian MCT1-MCT4, but only for MCT1 and MCT2 have detailed analyses of substrate and inhibitor kinetics been described following heterologous expression in Xenopus oocytes. MCT1 is ubiquitously expressed, but is especially prominent in heart and red muscle, where it is up-regulated in response to increased work, suggesting a special role in lactic acid oxidation. By contrast, MCT4 is most evident in white muscle and other cells with a high glycolytic rate, such as tumour cells and white blood cells, suggesting it is expressed where lactic acid efflux predominates. MCT2 has a ten-fold higher affinity for substrates than MCT1 and MCT4 and is found in cells where rapid uptake at low substrate concentrations may be required, including the proximal kidney tubules, neurons and sperm tails. MCT3 is uniquely expressed in the retinal pigment epithelium. The mechanisms involved in regulating the expression of different MCT isoforms remain to be established. However, there is evidence for alternative splicing of the 5'- and 3'-untranslated regions and the use of alternative promoters for some isoforms. In addition, MCT1 and MCT4 have been shown to interact specifically with OX-47 (CD147), a member of the immunoglobulin superfamily with a single transmembrane helix. This interaction appears to assist MCT expression at the cell surface. There is still much work to be done to characterize the properties of the different isoforms and their regulation, which may have wide-ranging implications for health and disease. In the future it will be interesting to explore the linkage of genetic diseases to particular MCTs through their chromosomal location.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Prótons , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/genética , Evolução Molecular , Humanos , Dados de Sequência Molecular , Transportadores de Ácidos Monocarboxílicos , Especificidade de Órgãos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Relação Estrutura-AtividadeRESUMO
Mitochondria from different regions of the brain were prepared, and the activation of the mitochondrial permeability transition (MPT) by calcium was investigated by monitoring the associated mitochondrial swelling. In general, the properties of the MPT in brain mitochondria were found to be qualitatively similar to those observed in liver and heart mitochondria. Thus, swelling was inhibited by adenine nucleotides (AdNs) and low pH (<7.0), whereas thiol reagents and alkalosis facilitated swelling. Cyclosporin A and its nonimmunosuppressive analogue N-methyl-Val-4-cyclosporin A (PKF 220-384) both inhibited swelling and prevented the translocation of cyclophilin D from the matrix to the membranes of cortical mitochondria. However, the calcium sensitivity of the MPT differed in mitochondria from three brain regions (hippocampus > cortex > cerebellum) and is correlated with the susceptibility of these regions to ischemic damage. Depleting mitochondria of AdNs by treatment with pyrophosphate ions sensitized the MPT to [Ca2+] and abolished regional differences, implying regional differences in mitochondrial AdN content. This was confirmed by measurements showing significant differences in AdN content among regions (cerebellum > cortex > hippocampus). Our data add to recent evidence that the MPT may be involved in neuronal death.
Assuntos
Nucleotídeos de Adenina/metabolismo , Encéfalo/fisiologia , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Animais , Encéfalo/ultraestrutura , Cerebelo/fisiologia , Córtex Cerebral/fisiologia , Ciclosporina/farmacologia , Ácido Egtázico/farmacologia , Hipocampo/fisiologia , Concentração de Íons de Hidrogênio , Membranas Intracelulares/fisiologia , Masculino , Mitocôndrias/efeitos dos fármacos , Dilatação Mitocondrial , Especificidade de Órgãos , Consumo de Oxigênio , Permeabilidade , Ratos , Ratos WistarRESUMO
The profiles of the lactate/H+ transporter isoforms [monocarboxylate transporter isoforms (MCT)] MCT1 and MCT4 (formerly MCT3 of Price, N. T., V. N. Jackson, and A. P. Halestrap. Biochem. J. 329: 321-328, 1998) were studied in the soleus, triceps brachii, and vastus lateralis muscles of six male subjects. The fiber-type compositions of the muscles were evaluated from the occurrence of the myosin heavy chain isoforms, and the fibers were classified as type I, IIA, or IIX. The total content of MCT1 and MCT4 was determined in muscle homogenates by Western blotting, and MCT1 and MCT4 were visualized on cross-sectional muscle sections by immunofluorescence microscopy. The Western blotting revealed a positive, linear relationship between the MCT1 content and the occurrence of type I fibers in the muscle, but no significant relation was found between MCT4 content and fiber type. Moreover, the interindividual variation in MCT4 content was much larger than the interindividual variation in MCT1 content in homogenate samples. The immunofluorescence microscopy showed that within a given muscle section, the MCT4 isoform was clearly more abundant in type II fibers than in type I fibers, whereas only minor differences existed in the occurrence of the MCT1 isoform between type I and II fibers. Together the present results indicate that the content of MCT1 in a muscle varies between different muscles, whereas fiber-type differences in MCT1 content are minor within a given muscle section. In contrast, the content of MCT4 is clearly fiber-type specific but apparently quite similar in various muscles.
Assuntos
Proteínas de Transporte/análise , Músculo Esquelético/química , Adulto , Biópsia por Agulha , Western Blotting , Imunofluorescência , Humanos , Masculino , Microscopia de Fluorescência , Transportadores de Ácidos Monocarboxílicos , Fibras Musculares Esqueléticas/química , Distribuição TecidualRESUMO
We have used mitochondrial entrapment of 2-deoxy-D-[3H]glucose (2-DG) to demonstrate that recovery of Langendorff-perfused rat hearts from ischemia is accompanied by reversal of the mitochondrial permeability transition (MPT). In hearts loaded with 2-DG before 40 min of ischemia and 25 min of reperfusion, 2-DG entrapment [expressed as 10(5) x (mitochondrial 2-[3H]DG dpm per unit citrate synthase)/(total heart 2-[3H]DG dpm/g wet wt)] increased from 11.1 +/- 1.3 (no ischemia, n = 4) to 32.5 +/- 1.9 (n = 6; P < 0.001). In other experiments, 2-DG was loaded after 25 min of reperfusion to determine whether some mitochondria that had undergone the MPT during the initial phase of reperfusion subsequently "resealed" and thus no longer took up 2-DG. The reduction of 2-DG entrapment to 20. 6 +/- 2.4 units (n = 5) confirmed that this was the case. Pyruvate (10 mM) in the perfusion medium increased recovery of left ventricular developed pressure from 57.2 +/- 10.3 to 98.9 +/- 10.8% (n = 6; P < 0.05) and reduced entrapment of 2-DG loaded preischemically and postischemically to 23.5 +/- 1.5 (n = 4; P < 0. 001) and 10.5 +/- 0.5 (n = 4; P < 0.01) units, respectively. The presence of pyruvate increased tissue lactate content at the end of ischemia and decreased the effluent pH during the initial phase of reperfusion concomitant with an increase in lactate output. We suggest that pyruvate may inhibit the MPT by decreasing pHi and scavenging free radicals, thus protecting hearts from reperfusion injury.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Isquemia Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Ácido Pirúvico/farmacologia , Nucleotídeos de Adenina/metabolismo , Animais , Desoxiglucose/farmacocinética , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ácido Láctico/metabolismo , Masculino , Mitocôndrias Cardíacas/fisiologia , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Concentração Osmolar , Ratos , Ratos WistarRESUMO
First, we present a summary of the evidence for our model of the molecular mechanism of the permeability transition (MPT). Our proposal is that the MPT occurs as a result of the binding of mitochondrial cyclophilin (CyP-D) to the adenine nucleotide translocase (ANT) in the inner mitochondrial membrane. This binding is enhanced by thiol modification of the ANT caused by oxidative stress or other thiol reagents. CyP-D binding enhances the ability of the ANT to undergo a conformational change triggered by Ca2+. Binding of ADP or ATP to a matrix site of the ANT antagonises this effect of Ca2+; modification of other ANT thiol groups inhibits ADP binding and sensitises the MPT to [Ca2+]. Increased membrane potential changes the ANT conformation to enhance ATP binding and hence inhibit the MPT. Our most recent data shows that a fusion protein of CyP-D and glutathione-S-transferase immobilised to Sepharose specifically binds the ANT from Triton-solubilised inner mitochondrial membranes in a cyclosporin A (CsA) sensitive manner. Second we summarise the evidence for the MPT being a major factor in the transition from reversible to irreversible injury during reperfusion of a heart following a period of ischaemia. We describe how in the perfused heart [3H]deoxyglucose entrapment within mitochondria can be used to measure the opening of MPT pore in situ. During ischaemia pore opening does not occur, but significant opening does occur during reperfusion, and recovery of the heart is dependent on subsequent pore closure. Pore opening is inhibited by the presence in the perfusion medium of pyruvate and the anaesthetic propofol which both protect the heart from reperfusion injury. Third we discuss how the MPT may be involved in determining whether cell death occurs by necrosis (extensive pore opening and ATP depletion) or apoptosis (transient pore opening with maintenance of ATP).
Assuntos
Canais de Cálcio/metabolismo , Mitocôndrias Cardíacas/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Cálcio/antagonistas & inibidores , Cálcio/metabolismo , Morte Celular/fisiologia , Desoxiglucose/metabolismo , Sequestradores de Radicais Livres/farmacologia , Coração/fisiopatologia , Humanos , Mitocôndrias Cardíacas/ultraestrutura , Translocases Mitocondriais de ADP e ATP/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Necrose , Peptidilprolil Isomerase/metabolismo , Perfusão , Permeabilidade , Propofol/farmacologiaRESUMO
The newly cloned proton-linked monocarboxylate transporter MCT3 was shown by Western blotting and immunofluorescence confocal microscopy to be expressed in all muscle fibers. In contrast, MCT1 is expressed most abundantly in oxidative fibers but is almost totally absent in fast-twitch glycolytic fibers. Thus MCT3 appears to be the major MCT isoform responsible for efflux of glycolytically derived lactic acid from white skeletal muscle. MCT3 is also expressed in several other tissues requiring rapid lactic acid efflux. The expression of both MCT3 and MCT1 was decreased by 40-60% 3 weeks after denervation of rat hind limb muscles, whereas chronic stimulation of the muscles for 7 days increased expression of MCT1 2-3-fold but had no effect on MCT3 expression. The kinetics and substrate and inhibitor specificities of monocarboxylate transport into cell lines expressing only MCT3 or MCT1 have been determined. Differences in the properties of MCT1 and MCT3 are relatively modest, suggesting that the significance of the two isoforms may be related to their regulation rather than their intrinsic properties.
Assuntos
Proteínas de Transporte/metabolismo , Ácido Láctico/metabolismo , Músculo Esquelético/metabolismo , Animais , Células COS , Catálise , Bovinos , Cinética , Proteínas de Membrana Transportadoras , Transportadores de Ácidos Monocarboxílicos , Denervação Muscular , Músculo Esquelético/inervação , Ratos , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
In vivo, butyrate is a major energy source for the colonic epithelium and is thought to stimulate proliferation. In contrast, butyrate in vitro has been shown to inhibit proliferation and induce differentiation and apoptosis in colonic epithelial cells. Most colon cell cultures are grown in medium containing high concentrations of glucose, whereas in vivo, the main energy source used by the colon cells is butyrate. The aim of this study was to determine whether the apparent contrasting roles of butyrate in vivo and in vitro could be as a consequence of differences in glucose availability. The sensitivity of two human colorectal tumour cell lines, one adenoma (S/RG/C2) and one carcinoma (HT29) to butyrate-induced growth inhibition and apoptosis was investigated to determine whether these cellular effects were altered under glucose depleted culture conditions. Glucose depletion resulted in increased apoptosis in both cell lines in the absence of butyrate. Butyrate in standard culture conditions (containing 25 mM glucose and 1 mM pyruvate) inhibited growth and induced apoptosis in both cell lines. However, low concentrations of butyrate in glucose depleted culture conditions (i.e. standard growth medium without glucose and pyruvate supplements) were found to reduce apoptosis induced by glucose deprivation and increase cell yield in both cell lines. The results show that in glucose depleted culture conditions, butyrate at low concentrations (0.5 mM for S/RG/C2, and 0.5 and 2 mM for HT29 cells) was found to be growth stimulatory whereas in the presence of glucose, these same concentrations of butyrate induced apoptosis. Thus, whether butyrate is growth stimulatory or growth inhibitory may depend on the availability of other energy sources. These observations may, in part, provide an explanation for the apparent opposite effects of butyrate on proliferation reported in vivo and in vitro.
Assuntos
Apoptose/efeitos dos fármacos , Butiratos/farmacologia , Colo/citologia , Colo/efeitos dos fármacos , Adenoma/metabolismo , Butiratos/metabolismo , Ácido Butírico , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Colo/metabolismo , Neoplasias do Colo/metabolismo , Meios de Cultura , Metabolismo Energético , Células Epiteliais , Epitélio/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Células HT29/citologia , Células HT29/efeitos dos fármacos , Humanos , Ácido Pirúvico/metabolismo , Ácido Pirúvico/farmacologia , Estimulação QuímicaRESUMO
Treatment of intact erythrocytes with 4,4'-diisothiocyanostilbene-2, 2'-disulfonate (DIDS) causes irreversible inhibition and chemical labeling of the lactate transporter, monocarboxylate transporter 1 (MCT1) (Poole, R. C., and Halestrap, A. P. (1992) Biochem. J. 283, 855-862). In rat erythrocytes DIDS also causes cross-linking of MCT1 to another protein in the membrane to give a product of 130 kDa on SDS-polyacrylamide gel electrophoresis. Cross-linking is markedly reduced by those compounds that protect against irreversible inhibition of lactate transport by DIDS and enhanced by imposition of a pH gradient across the plasma membrane to recruit the substrate binding site of MCT1 to an exofacial conformation. These data indicate that DIDS cross-linking is via the same site on MCT1 as is responsible for inhibition of transport. Antibodies raised against the cross-linked conjugate react with proteins of approximately 40 kDa (MCT1) and 70 kDa on Western blots of erythrocyte membranes and an additional band of 130 kDa after treatment of erythrocytes with 100 microM DIDS. The 70-kDa protein that is cross-linked to MCT1 was purified and shown to contain N-linked carbohydrate; the apparent core molecular mass is 40 kDa. Amino acid sequencing showed that the protein is the rat equivalent of the membrane-spanning mouse teratocarcinoma glycoprotein GP-70, a member of the immunoglobulin superfamily related to basigin (Ozawa, M., Huang, R. P., Furukawa, T. , and Muramatsu, T. (1988) J. Biol. Chem. 263, 3059-3062). Possible implications of the specific interaction between MCT1 and this protein are discussed.
Assuntos
Proteínas de Transporte/sangue , Membrana Eritrocítica/metabolismo , Glicoproteínas de Membrana/sangue , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Transporte/efeitos dos fármacos , Reagentes de Ligações Cruzadas , Dimerização , Imunoglobulinas/química , Cinética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Peso Molecular , Transportadores de Ácidos Monocarboxílicos , Fragmentos de Peptídeos/química , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Teratoma , TripsinaRESUMO
We have investigated cell metabolism during apoptosis in the murine interleukin-3 (IL-3)-dependent cell line Bo and two derivative clones (B14 and B15) overexpressing human bcl-2a. On removal of IL-3, Bo cells underwent apoptosis within 8 h, whereas B14 and B15 cells were resistant for at least 24 h. Metabolically, Bo, B14, and B15 cells were indistinguishable from each other. All were insensitive to mitochondrial poisons, derived ATP entirely by glycolysis, and maintained similar mitochondrial membrane potentials measured by rhodamine-123 fluorescence with or without IL-3. All virtually ceased glycolysis and production of lactic acid on IL-3 withdrawal but maintained intracellular [ATP] until in Bo cultures the cells began to apoptose. B14 and B15 cells became glycolytically arrested but maintained stable ATP levels during protection from apoptosis. Depletion of intracellular ATP by uncoupling the mitochondrial ATPase with 2,4-dinitrophenol or carbonyl cyanide p-trifluoromethoxyphenylhydrazone induced apoptosis in Bo cells with or without IL-3, but not in B14 or B15 cells. bcl-2-overexpressing cells were recoverable with high plating efficiency even after prolonged exposure to 2,4-dinitrophenol. We conclude that IL-3 withdrawal leads to arrest of energy metabolism in which ATP levels are maintained. In Bo cells this is followed by apoptosis, whereas in bcl-2-overexpressing cells this state is stably prolonged. ATP depletion is a strong apoptotic signal which overrides IL-3 signaling in normal cells but is ineffective in bcl-2-overexpressing cells. Prolonged metabolic arrest and resistance to ATP depletion facilitated by bcl-2 are both reversible. Persistent reversible metabolic dormancy would provide cells with a survival advantage in nonsustainable environments (e.g. hypoxia or substrate lack) and suggests a mechanism for the survival advantage displayed by cells overexpressing bcl-2.