Inhibiting mitochondrial permeability transition pore opening: a new paradigm for myocardial preconditioning?
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OBJECTIVE: We propose that ischemic preconditioning (IPC) and mitochondrial K(ATP) channel activation protect the myocardium by inhibiting mitochondrial permeability transition pore (MPTP) opening at reperfusion. METHODS: Isolated rat hearts were subjected to 35 min ischemia/120 min reperfusion and assigned to the following groups: (1) control; (2) IPC of 2x5 min each of preceding global ischemia; (3,4,5) 0.2 micromol/l cyclosporin A (CsA, which inhibits MPTP opening), 5 micromol/l FK506 (which inhibits the phosphatase calcineurin without inhibiting MPTP opening), or 20 micromol/l atractyloside (Atr, a MPTP opener) given at reperfusion; (6,7) pre-treatment with 30 micromol/l diazoxide (Diaz, a mitochondrial K(ATP) channel opener) or 200 nmol/l 2 chloro-N(6)-cyclopentyl-adenosine (CCPA, an adenosine A1 receptor agonist); (8) IPC+Atr; (9) Diaz+Atr; (10) CCPA+Atr. The effect of mitochondrial K(ATP) channel activation on calcium-induced MPTP opening in isolated calcein-loaded mitochondria was also assessed. RESULTS: IPC, CsA when given at reperfusion, and pre-treatment with diazoxide or CCPA all limited infarct size (19.9+/-2.6% in IPC; 24.6+/-1.9% in CsA, 18.0+/-1.7% in Diaz, 20.4+/-3.3% in CCPA vs. 44.7+/-2.0% in control, P<0.0001). Opening the MPTP with atractyloside at reperfusion abolished this cardio-protective effect (47.7+/-1.8% in IPC+Atr, 42.3+/-3.2% in Diaz+Atr, 51.2+/-1.6% in CCPA+Atr). Atractyloside and FK506, given at reperfusion, did not influence infarct size (45.7+/-2.1% in Atr and 43.1+/-3.6% in FK506 vs. 44.7+/-2.0% in control, P=NS). Diazoxide (30 micromol/l) was shown to reduce calcium-induced MPTP opening by 52.5+/-8.0% in calcein-loaded mitochondria. 5-Hydroxydecanoic acid (100 micromol/l) was able to abolish the cardio-protective effects of both diazoxide and IPC. CONCLUSION: One interpretation of these data is that IPC and mitochondrial K(ATP) channel activation may protect the myocardium by inhibiting MPTP opening at reperfusion. (+info)
Control of pyruvate dehydrogenase activity in intact cardiac mitochondria. Regulation of the inactivation and activation of the dehydrogenase.
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The control of pyruvate dehydrogenase activity by inactivation and activation was studied in intact mitochondria isolated from rabbit heart. Pyruvate dehydrogenase could be completely inactivated by incubating mitochondria with ATP, oligomycin, and NaF. This loss in dehydrogenase activity was correlated with the incorporation of 32P from [gamma-32P]ATP into mitochondrial protein(s) and with a decrease in the mitochondrial oxidation of pyruvate. ATP may be supplied exogenously, generated from endogenous ADP during oxidative phosphorylation, or formed from exogenous ADP in carbonyl cyanid p-trifluoromethoxyphenylhydrazone-uncoupled mitochondria. With coupled mitochondria the concentration of added ATP required to half-inactivate the dehydrogenase was 0.24 mM. With uncoupled mitochondria the apparent Km was decreased to 60 muM ATP. Inactivation of pyruvate dehydrogenase by exogenous ATP was sensitive to atractyloside, suggesting that pyruvate dehydrogenase kinase acts internally to the atractyloside-sensitive barrier. The divalent cation ionophore, A23187, enhanced the loss of dehydrogenase activity. Pyruvate dehydrogenase activity is regulated additionally by pyruvate, inorganic phosphate, and ADP. Pyruvate, in the presence of rotenone, strongly inhibited inactivation. This suggests that pyruvate facilitates its own oxidation and that increases in pyruvate dehydrogenase activity by substrate may provide a modulating influence on the utilization of pyruvate via the tricarboxylate cycle. Inorganic phosphate protected the dehydrogenase from inactivation by ATP. ADP added to the incubation mixture together with ATP inhibited the inactivation of pyruvate dehydrogenase. This protection may result from a direct action on pyruvate dehydrogenase kinase, as ADP competes with ATP, and an indirect action, in that ADP competes with ATP for the translocase. It is suggested that the intramitochondrial [ATP]:[ADP] ratio effects the kinase activity directly, whereas the cytosolic [ATP]:[ADP] ratio acts indirectly. Mg2+ enhances the rate of reactivation of the inactivated pyruvate dehydrogenase presumably by accelerating the rate of dephosphorylation of the enzyme. Maximal activation is obtained with the addition of 0.5 mM Mg2+.. (+info)
Hamster brown-adipose-tissue mitochondria. Purine nucleotide control of the ion conductance of the inner membrane, the nature of the nucleotide binding site.
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The inner membrane of hamster brown adipose tissue mitochondria possesses a mechanism for the conductance of protons (or hydroxyl ions) and halide anions which may be specifically inhibited by exogenous purine nucleoside di- or triphosphates. The mechanism of the nucleotide interaction is examined. The added nucleotides can inhibit the ion conductances without equilibrating with the matrix pools of purine nucleotides. ADP translocation is completely sensitive to atractylate, and no mechanism for GDP translocation could be detected. The nucleotides act on the conductance mechanism without covalent modification. A purine nucleotide binding site is described which is distinct from the adenine nucleotide translocase, does not bind atractylate, has a capacity of 0.7 nmol - mg-1, and affinities, specificities and a pH dependency closely corresponding to the conditions required for the inhibition of the ion conductances. The binding site is not apparent in rat liver mitochondria. A causal relationship is suggested between the occupation of this site by added purine nucleotides, and the inhibition of the ion conductance pathway. The role of the pathway in the physiological control of non-shivering thermogenesis by the tissue is discussed. (+info)
Mode of stimulation by aldosterone of the sodium efflux in barnacle muscle fibres: effects of ouabain, ethacrynic acid, diphenylhydantoin, (ATPMg)(2-), adenine translocase inhibitors, pyruvate and oxythiamine.
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1. A study has been made of the nature of the delayed stimulation caused by external aldosterone in barnacle fibres pre-exposed to aldosterone. 2. (i) Microinjection of 0-5 M-ATPMg2- caused only a small but prompt rise in the Na efflux. (ii) Microinjection of 0-5 M-ATPMg2- followed by external application of 10(-5)M aldosterone greatly augmented the magnitude of the delayed stimulation. The response was dose-dependent, as well as dependent on the concentration of external K+ and H+, but not Na+, Ca2+ or Mg2+. (iii) External application of 10(-5) M aldosterone for 30 min followed by its withdrawal from the bathing medium failed to bring about delayed stimulation. By contrast, fibres into which ATP had been injected showed delayed stimulation under these conditions. 3. Microinjection of actinomycin-D or spironolactone SC-14266 into fibres into which ATP had been injected followed by external application of aldosterone resulted in complete abolition of the delayed stimulation. 4. Delayed stimulation was reduced whether ATP had been injected or not by prior external application of 10(-4)M ouabain or internal application of 8 x 10(-2)M ethacrynic acid. It was completely abolished by prior application of ouabain externally and ethacrynic acid internally, or only 10(-4)M diphenylhydantoin externally. 5. (i) Microinjection of atractyloside or bongkrekic acid caused a substantial fall in the resting Na efflux. Bonkrekic acid proved more powerful than atractyloside. Microinjection of 0-05 M-ATPMg2- into fibres poisoned with 2-0 x 10(-2)M bongkrekic acid completely restored the Na efflux. (+info)
Comparison of the effect of mitochondrial inhibitors on mitochondrial membrane potential in two different cell lines using flow cytometry and spectrofluorometry.
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BACKGROUND: Determination of mitochondrial membrane potential (DeltaPsim) is widely used to characterize cellular metabolism, viability, and apoptosis. Changes of DeltaPsim induced by inhibitors of oxidative phosphorylation characterize respective contributions of mitochondria and glycolysis to adenosine triphosphate (ATP) synthesis. METHODS: DeltaPsim in BSC-40 and HeLa G cell lines was determined by flow cytometry and spectrofluorometry. Its changes induced by specific mitochondrial inhibitors were evaluated using 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)), tetramethylrhodamine ethyl ester, and MitoTracker Red. Mitochondrial function was further characterized by oxygen consumption. RESULTS: Inhibition of respiration by antimycin A or uncoupling of mitochondria by FCCP decreased DeltaPsim in both cell lines. Inhibition of ATP production by oligomycin or atractyloside induced a moderate decrease of DeltaPsim in HeLa G cells and an increase of DeltaPsim in BSC-40 cells. Statistically significant differences in DeltaPsim between the two cell lines were found with both flow cytometry and spectrofluorometry. Respirometry showed higher basal and FCCP-stimulated respiration in BSC-40 cells. CONCLUSION: Changes of DeltaPsim and oxygen consumption showed that BSC-40 cells are more sensitive than HeLa G cells to inhibitors of mitochondrial function, suggesting that BSC-40 cells are more dependent than HeLa G cells on aerobic ATP production. Determination of DeltaPsim changes by flow cytometry exhibited greater sensitivity than the ones by spectrofluorometry. (+info)
Protein kinase Cepsilon interacts with and inhibits the permeability transition pore in cardiac mitochondria.
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Although functional coupling between protein kinase Cepsilon (PKCepsilon) and mitochondria has been implicated in the genesis of cardioprotection, the signal transduction mechanisms that enable this link and the identities of the mitochondrial proteins modulated by PKCepsilon remain unknown. Based on recent evidence that the mitochondrial permeability transition pore may be involved in ischemia/reperfusion injury, we hypothesized that protein-protein interactions between PKCepsilon and mitochondrial pore components may serve as a signaling mechanism to modulate pore function and thus engender cardioprotection. Coimmunoprecipitation and GST-based affinity pull-down from mouse cardiac mitochondria revealed interaction of PKCepsilon with components of the pore, namely voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and hexokinase II (HKII). VDAC1, ANT1, and HKII were present in the PKCepsilon complex at approximately 2%, approximately 0.2%, and approximately 1% of their total expression, respectively. Moreover, in vitro studies demonstrated that PKCepsilon can directly bind and phosphorylate VDAC1. Incubation of isolated cardiac mitochondria with recombinant PKCepsilon resulted in a significant inhibition of Ca2+-induced mitochondrial swelling, an index of pore opening. Furthermore, cardiac-specific expression of active PKCepsilon in mice, which is cardioprotective, greatly increased interaction of PKCepsilon with the pore components and inhibited Ca2+-induced pore opening. In contrast, cardiac expression of kinase-inactive PKCepsilon did not affect pore opening. Finally, administration of the pore opener atractyloside significantly attenuated the infarct-sparing effect of PKCepsilon transgenesis. Collectively, these data demonstrate that PKCepsilon forms physical interactions with components of the cardiac mitochondrial pore. This in turn inhibits the pathological function of the pore and contributes to PKCepsilon-induced cardioprotection. (+info)
Carvedilol: relation between antioxidant activity and inhibition of the mitochondrial permeability transition.
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OBJECTIVES: The mitochondrial permeability transition (MPT) is an event related to severe oxidative stress (for example, during myocardial ischemia and reperfusion) and excessive mitochondrial calcium accumulation, also being implicated in cell death. In this study, we compared the effect of carvedilol on the cardiac MPT induced by calcium and phosphate (Ca/Pi) and calcium/carboxyatractyloside (Ca/Catr). Oxidative stress plays a major role in MPT induction by Ca/Pi, leading to the oxidation of protein thiol groups, in contrast with Ca/Catr, where such oxidation is secondary to MPT induction and is not caused by oxidative stress. MATERIALS AND METHODS: Mitochondria were isolated from rat hearts and parameters related to MPT induction were evaluated (n = 5 for each inducer): mitochondrial swelling and oxidation of protein thiol groups (both measured by spectrophotometry). RESULTS: Using Ca/Pi, carvedilol protected mitochondria from MPT induction, particularly in its high conductance form. Its effect was demonstrated by analyzing the decrease in mitochondrial swelling amplitude. Simultaneously, we observed inhibition of protein thiol group oxidation (p < 0.001). By contrast, carvedilol did not show any protective effect with Ca/Catr. CONCLUSIONS: Carvedilol was only effective against the MPT when the oxidation of protein thiol groups was the cause and not the consequence of the MPT phenomenon. The results clearly show that during myocardial aggressions (ischemia and reperfusion, for example), the protective effect of carvedilol is primarily due to an antioxidant mechanism, inhibiting the production and effects of reactive oxygen species. (+info)
A hallmark of central nervous system (CNS) pathology is reactive astrocyte production of the chronic glial scar that is inhibitory to neuronal regeneration. The reactive astrocyte response is complex; these cells also produce neurotrophic factors and are responsible for removal of extracellular glutamate, the excitatory neurotransmitter that rises to neurotoxic levels in injury and disease. To identify genes expressed by reactive astrocytes, we employed an in vivo model of the glial scar and differential display PCR and found an increase in the level of Ant1, a mitochondrial ATP/ADP exchanger that facilitates the flux of ATP out of the mitochondria. Ant1 expression in reactive astrocytes is regulated by transforming growth factor-beta1, a pluripotent CNS injury-induced cytokine. The significance of increased Ant1 is evident from the observation that glutamate uptake is significantly decreased in astrocytes from Ant1 null mutant mice while a specific Ant inhibitor reduces glutamate uptake in wild-type astrocytes. Thus, the astrocytic response to CNS injury includes an apparent increase in energy mobilization capacity by Ant1 that contributes to neuroprotective, energy-dependent glutamate uptake. (+info)