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1.
Br J Surg ; 106(1): 120-131, 2019 01.
Article in English | MEDLINE | ID: mdl-30259964

ABSTRACT

BACKGROUND: Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) is a two-stage strategy to induce rapid regeneration of the remnant liver. The technique has been associated with high mortality and morbidity rates. This study aimed to evaluate mitochondrial function, biogenesis and morphology during ALPPS-induced liver regeneration. METHODS: Male Wistar rats (n = 100) underwent portal vein ligation (PVL) or ALPPS. The animals were killed at 0 h (without operation), and 24, 48, 72 or 168 h after intervention. Regeneration rate and proliferation index were assessed. Mitochondrial oxygen consumption and adenosine 5'-triphosphate (ATP) production were measured. Mitochondrial biogenesis was evaluated by protein level measurements of peroxisome proliferator-activated receptor γ co-activator (PGC) 1-α, nuclear respiratory factor (NRF) 1 and 2, and mitochondrial transcription factor α. Mitochondrial morphology was evaluated by electron microscopy. RESULTS: Regeneration rate and Ki-67 index were significantly raised in the ALPPS group compared with the PVL group (regeneration rate at 168 h: mean(s.d.) 291·2(21·4) versus 245·1(13·8) per cent, P < 0·001; Ki-67 index at 24 h: 86·9(4·6) versus 66·2(4·9) per cent, P < 0·001). In the ALPPS group, mitochondrial function was impaired 48 h after the intervention compared with that in the PVL group (induced ATP production); (complex I: 361·9(72·3) versus 629·7(165·8) nmol per min per mg, P = 0·038; complex II: 517·5(48·8) versus 794·8(170·4) nmol per min per mg, P = 0·044). Markers of mitochondrial biogenesis were significantly lower 48 and 72 h after ALPPS compared with PVL (PGC1-α at 48 h: 0·61-fold decrease, P = 0·045; NRF1 at 48 h: 0·48-fold decrease, P = 0·028). Mitochondrial size decreased significantly after ALPPS (0·26(0·05) versus 0·40(0·07) µm2 ; P = 0·034). CONCLUSION: Impaired mitochondrial function and biogenesis, along with the rapid energy-demanding cell proliferation, may cause hepatocyte dysfunction after ALPPS. Surgical relevance Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) is a well known surgical strategy that combines liver partition and portal vein ligation. This method induces immense regeneration in the future liver remnant. The rapid volume increase is of benefit for resectability, but the mortality and morbidity rates of ALPPS are strikingly high. Moreover, lagging functional recovery of the remnant liver has been reported recently. In this translational study, ALPPS caused an overwhelming inflammatory response that interfered with the peroxisome proliferator-activated receptor γ co-activator 1-α-coordinated, stress-induced, mitochondrial biogenesis pathway. This resulted in the accumulation of immature and malfunctioning mitochondria in hepatocytes during the early phase of liver regeneration (bioenergetic destabilization). These findings might explain some of the high morbidity if confirmed in patients.


Subject(s)
Liver Regeneration/physiology , Mitochondria, Liver/physiology , Portal Vein/surgery , Adenosine Triphosphate/biosynthesis , Animals , Cell Proliferation/physiology , Hepatectomy/methods , Hepatocytes/physiology , Ligation/methods , Male , Microscopy, Electron , NADP/metabolism , Organelle Biogenesis , Oxygen Consumption/physiology , Rats, Wistar
2.
Neuropharmacology ; 123: 287-298, 2017 Sep 01.
Article in English | MEDLINE | ID: mdl-28495375

ABSTRACT

Methylene blue (MB), a potential neuroprotective agent, is efficient in various neurodegenerative disease models. Beneficial effects of MB have been attributed to improvements in mitochondrial functions. Substrate-level phosphorylation (SLP) results in the production of ATP independent from the ATP synthase (ATP-ase). In energetically compromised mitochondria, ATP produced by SLP can prevent the reversal of the adenine nucleotide translocase and thus the hydrolysis of glycolytic ATP. The aim of the present study was to investigate the effect of MB on mitochondrial SLP catalysed by succinyl-CoA ligase. Measurements were carried out on isolated guinea pig cortical mitochondria respiring on α-ketoglutarate, glutamate, malate or succinate. The mitochondrial functions and parameters like ATP synthesis, oxygen consumption, membrane potential, and NAD(P)H level were followed online, in parallel with the redox state of MB. SLP-mediated ATP synthesis was measured in the presence of inhibitors for ATP-ase and adenylate kinase. In the presence of the ATP-ase inhibitor oligomycin MB stimulated respiration with all of the respiratory substrates. However, the rate of ATP synthesis increased only with substrates α-ketoglutarate and glutamate (forming succinyl-CoA). MB efficiently stimulated SLP and restored the membrane potential in mitochondria also with the combined inhibition of Complex I and ATP synthase. ATP formed by SLP alleviated the energetic insufficiency generated by the lack of oxidative phosphorylation. Thus, the MB-mediated stimulation of SLP might be important in maintaining the energetic competence of mitochondria and in preventing the mitochondrial hydrolysis of glycolytic ATP. The mitochondrial effects of MB are explained by the ability to accept electrons from reducing equivalents and transfer them to cytochrome c bypassing the respiratory Complexes I and III.


Subject(s)
Acyl Coenzyme A/metabolism , Central Nervous System Agents/pharmacology , Citric Acid Cycle/drug effects , Methylene Blue/pharmacology , Mitochondria/drug effects , Adenosine Triphosphate/metabolism , Animals , Catalysis/drug effects , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Citric Acid Cycle/physiology , Guinea Pigs , Hydrolysis/drug effects , Male , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/physiology , Mitochondria/metabolism , NADP/metabolism , Oxygen Consumption/drug effects , Phosphorylation/drug effects
3.
Methods Enzymol ; 547: 199-223, 2014.
Article in English | MEDLINE | ID: mdl-25416360

ABSTRACT

In this chapter, we describe the currently most advanced methods applied for the quantitative assessment of ROS homeostasis inside the mitochondrion. These techniques are of particular interest in the field of oxidative stress. After discussing the importance of quantifying mitochondrial ROS homeostasis, three major aspects of this phenomenon and the pertinent methodologies for detection are delineated in detail. First the most important methods, based on fluorimetric or spectrophotometric approaches, for the detection of mitochondrial ROS are described. Elimination of ROS generated inside the mitochondrion is another crucial mechanism that also needs to be quantified accurately to estimate the antioxidant capacity of mitochondria under specific conditions. Since ROS generation and elimination manifest in concert, there needs to exist independent methods for the estimation of the net effect. Such a sensitive biochemical marker in the mitochondrion is aconitase, a citric acid cycle enzyme which is greatly sensitive to ROS. We describe two procedures for the precise determination of aconitase activity. A few auxiliary techniques and good practices having relevance in the successful accomplishment of the more delicate approaches are also mentioned. All other relevant technical considerations including advantages/disadvantages of the various methods and the most common artifacts are also discussed.


Subject(s)
Biochemistry/methods , Mitochondria/metabolism , Reactive Oxygen Species/analysis , Reactive Oxygen Species/metabolism , Aconitate Hydratase/metabolism , Animals , Cytochromes c/metabolism , Homeostasis , Homovanillic Acid/metabolism , Humans , Hydrogen Peroxide/analysis , Hydrogen Peroxide/metabolism , Membrane Potential, Mitochondrial , Mitochondrial Proteins/metabolism , Oxidative Stress , Spectrometry, Fluorescence/methods
4.
Free Radic Biol Med ; 77: 317-30, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25277417

ABSTRACT

The redox dye methylene blue (MB) is proven to have beneficial effects in various models of neurodegenerative diseases. Here we investigated the effects of MB (100 nM, 300 nM, and 1 µM) on key bioenergetic parameters and on H2O2 production/elimination in isolated guinea pig brain mitochondria under normal as well as respiration-impaired conditions. As measured by high-resolution Oxygraph the rate of resting oxygen consumption was increased, but the ADP-stimulated respiration was unaffected by MB with any of the substrates (glutamate malate, succinate, or α-glycerophosphate) used for supporting mitochondrial respiration. In mitochondria treated with inhibitors of complex I or complex III MB moderately but significantly increased the rate of ATP production, restored ΔΨm, and increased the rate of Ca(2+) uptake. The effects of MB are consistent with transferring electrons from upstream components of the electron transport chain to cytochrome c, which is energetically favorable when the flow of electrons in the respiratory chain is compromised. On the other hand, MB significantly increased the production of H2O2 measured by Amplex UltraRed fluorimetry under all conditions, in resting, ATP-synthesizing, and respiration-impaired mitochondria, with each substrate combination supporting respiration. Furthermore, it also decreased the elimination of H2O2. Generation of H2O2 without superoxide formation, observed in the presence of MB, is interpreted as a result of reduction of molecular oxygen to H2O2 by the reduced MB. The elevated generation and impaired elimination of H2O2 should be considered for the overall oxidative state of mitochondria treated with MB.


Subject(s)
Energy Metabolism/drug effects , Hydrogen Peroxide/metabolism , Methylene Blue/pharmacology , Mitochondria/metabolism , Neuroprotective Agents/pharmacology , Adenosine Triphosphate/biosynthesis , Animals , Calcium/metabolism , Drug Evaluation, Preclinical , Guinea Pigs , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Oxidation-Reduction , Oxygen Consumption/drug effects
5.
Free Radic Res ; 48(10): 1190-9, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24985354

ABSTRACT

Robust production of reactive oxygen species (ROS) by phagocyte NADPH oxidase (phox) during the respiratory burst (RB) is a characteristic feature of eosinophil and neutrophil granulocytes. In these cells the voltage-gated proton channel (Hv1) is now considered as an ancillary subunit of the phox needed for intense ROS production. Multiple sources reported that the expression of phox subunits and RB is more intensive in eosinophils than in neutrophils. In most of these studies the eosinophils were not isolated from healthy individuals, and a comparative analysis of Hv1 expression had never been carried out. We performed a systematic comparison of the levels of essential phox subunits, Hv1 expression and ROS producing capacity between eosinophils and neutrophils of healthy individuals. The expression of phox components was similar, whereas the amount of Hv1 was ∼ 10-fold greater in eosinophils. Furthermore, Hv1 expression correlated with Nox2 expression only in eosinophils. Additionally, in confocal microscopy experiments co-accumulation of Hv1 and Nox2 at the cell periphery was observed in resting eosinophils but not in neutrophils. While phorbol-12-myristate-13-acetate-induced peak extracellular ROS release was ∼ 1.7-fold greater in eosinophils, oxygen consumption studies indicated that the maximal intensity of the RB is only ∼ 1.4-fold greater in eosinophils. Our data reinforce that eosinophils, unlike neutrophils, generate ROS predominantly extracellularly. In contrast to previous works we have found that the two granulocyte types display very similar phox subunit expression and RB capacity. The large difference in Hv1 expression suggests that its support to intense ROS production is more important at the cell surface.


Subject(s)
Eosinophils/metabolism , Ion Channels/metabolism , Neutrophils/metabolism , Respiratory Burst/physiology , Fluorescent Antibody Technique , Humans , Immunoblotting , Membrane Glycoproteins/metabolism , Microscopy, Confocal , NADPH Oxidase 2 , NADPH Oxidases/metabolism , Oxygen Consumption/physiology , Reactive Oxygen Species/metabolism
6.
Neuroscience ; 120(3): 771-81, 2003.
Article in English | MEDLINE | ID: mdl-12895517

ABSTRACT

Brain ischemia is frequently associated with oxidative stress in the reperfusion period. It is known that noradrenaline (NA) is released in excess under energy deprivation by the sodium-dependent reversal of the monoamine carrier. However, it is not known how oxidative stress affects NA release in the brain alone or in combination with energy deprivation. As a model of oxidative stress, the effect of H(2)O(2) (0.1-1.5 mM) perfusion was investigated in superfused rat hippocampal slices. It elicited a dose-dependent elevation of the release of [(3)H]NA and its tritiated metabolites as well as a simultaneous drop in the tissue energy charge. Mitochondrial inhibitors, i.e. rotenone (10 microM), and oligomycin (10 microM) in combination, also decreased the energy charge, but they had only a mild effect on [(3)H]NA release. However, when H(2)O(2) was added together with oligomycin and rotenone their effect on [(3)H]NA release was greatly exacerbated. H(2)O(2) and mitochondrial inhibitors also induced an increase in [Na(+)](i) in isolated nerve terminals, and their effect was additive. The effect of H(2)O(2) on tritium release was temperature-dependent. It was also attenuated by the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (30 microM) and (+/-)-2-amino-5-phosphonopentanoic acid (10 microM), by the nitric oxide synthase inhibitors, N omega-nitro-L-arginine methyl ester (100 microM), or 7-nitroindazole (50 microM) and by the vesicular uptake inhibitor tetrabenazine (1 microM). Our results suggest that oxidative stress releases glutamate followed by activation of postsynaptic ionotropic glutamate receptors that trigger nitric oxide production and results in a flood of NA from cytoplasmic stores. The massive elevation of extracellular NA under conditions of oxidative stress combined with mitochondrial dysfunction may provide an additional source of highly reactive free radicals thus initiating a self-amplifying cycle leading to neuronal degeneration.


Subject(s)
Hippocampus/metabolism , Mitochondria/metabolism , Norepinephrine/metabolism , Oxidative Stress , Animals , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Excitatory Amino Acid Antagonists/pharmacology , Extracellular Space/metabolism , Glutamic Acid/metabolism , Hippocampus/drug effects , Hydrogen Peroxide/adverse effects , Male , Nitric Oxide/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Oligomycins/pharmacology , Presynaptic Terminals/metabolism , Rats , Rats, Wistar , Rotenone/pharmacology , Sodium/metabolism , Sodium Channel Blockers/pharmacology , Tetrodotoxin/pharmacology , Uncoupling Agents/pharmacology
7.
Neuroscience ; 104(3): 761-8, 2001.
Article in English | MEDLINE | ID: mdl-11440807

ABSTRACT

Our aim was to investigate the mechanisms involved in the high intracellular sodium-induced transmitter release in the CNS through the characterisation of the veratridine-evoked (40 microM) noradrenaline release from rat hippocampal slices. The response to veratridine was completely inhibited by tetrodotoxin (1 microM), indicating that the effect is due to the activation of sodium channels. Omission of Ca2+ from the superfusion fluid inhibited the veratridine-evoked release by 72%, showing that the majority of release results from external Ca2+-dependent exocytosis. The residual Ca2+-independent release was not blocked by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (100 microM) suggesting that intracellular Ca2+ stores are not involved in this component of veratridine effect. The noradrenaline uptake blockers, desipramine (10 microM) and nisoxetine (10 microM), inhibited the external Ca2+-independent release by 50 and 46%, respectively, indicating that the release partly originates from the reversal of transporters (carrier-mediated release). In contrast to uptake blockers, lowering the temperature, another possibility to inhibit transporter function, completely inhibited the effect of veratridine in the absence of Ca2+. Further experiments revealed that low temperature (20 and 12 degrees C) reduces the veratridine-induced increase of intracellular sodium concentration ([Na+]i) in rat cortical synaptosomes (68 and 78% inhibition, respectively). The clinical relevance of our data is that during ischemia a massive release of transmitters occurs mainly due to the elevation of [Na+]i, which contributes to the development of ischemic brain injury. Our results show that low temperature may be a better therapeutic approach to the treatment of ischemia because it has a dual action on this process. Firstly, it inhibits the function of uptake transporters and hence reduces the carrier-mediated outflow of transmitters. Secondly, it inhibits the sodium influx and therefore prevents the unwanted elevation of [Na+]i. Our data also suggest that veratridine stimulation can be a suitable model for ischemic conditions.


Subject(s)
Brain Ischemia/metabolism , Hippocampus/drug effects , Intracellular Fluid/drug effects , Neurons/drug effects , Norepinephrine/metabolism , Sodium Channels/drug effects , Sodium/metabolism , Adrenergic Uptake Inhibitors/pharmacology , Animals , Brain Ischemia/physiopathology , Brain Ischemia/therapy , Calcium/metabolism , Calcium Signaling/drug effects , Calcium Signaling/physiology , Chelating Agents/pharmacology , Exocytosis/drug effects , Exocytosis/physiology , Extracellular Space/drug effects , Extracellular Space/metabolism , Hippocampus/cytology , Hippocampus/metabolism , Hypothermia, Induced , Intracellular Fluid/metabolism , Male , Neurons/cytology , Neurons/metabolism , Norepinephrine/pharmacokinetics , Organ Culture Techniques , Rats , Rats, Sprague-Dawley , Sodium Channels/metabolism , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , Synaptic Vesicles/drug effects , Synaptic Vesicles/metabolism , Synaptosomes/drug effects , Synaptosomes/metabolism , Tritium , Veratridine/pharmacology
8.
Environ Toxicol Pharmacol ; 8(2): 83-94, 2000 Jan 01.
Article in English | MEDLINE | ID: mdl-10867367

ABSTRACT

In this report, evidence is presented that the marine unicellular eukaryotic dinoflagellates can cause neurotoxicity very likely by an increase in intracellular free calcium ions ([Ca(2+)](i)). Determinations of the effects of culture supernatants from different clones of the dinoflagellate Alexandrium sp. isolated from algal blooms on the viability of rat primary neuronal cells revealed that all clones tested were toxic for these cells. In addition, all Alexandrium clones tested, except for A. ostenfeldii BAH ME-141, were found to be toxic for rat pheochromocytoma PC12 cells. No toxicity was observed for culture supernatants from Gonyaulax and Coolia monotis. Calcium ions are important in the process of apoptotic cell death; our studies revealed that the dinoflagellate supernatants from A. lusitanicum K2, A. lusitanicum BAH ME-091, and A. tamarense 1M caused an increase in [Ca(2+)](i) levels in both PC12 cells and primary neuronal cells. These dinoflagellate supernatants, as well as the A. tamarense ccmp 115 supernatant, were found to cause also an increase in free calcium concentration in isolated synaptosomes. Our results suggest that the neurotoxic effects of certain dinoflagellate supernatants may be associated with disturbances in [Ca(2+)](i) levels.

9.
Neurochem Int ; 36(6): 483-8, 2000 May.
Article in English | MEDLINE | ID: mdl-10762084

ABSTRACT

We have reported recently (Chinopoulos et al., 1999 J. Neurochem. 73, 220 228) that mitochondrial membrane potential (delta(psi)m) in isolated nerve terminals is markedly reduced by H2O2 in the absence of F0F1-ATPase working as a proton pump. Here we demonstrate that delta(psi)m reduced by H2O2 (0.5 mM) in the presence of oligomycin (10 mM), an inhibitor of the F0F1-ATPase, was able to recover by the addition of catalase (2000 U). Similarly, a decrease in the NAD(P)H level due to H2O2 can be reversed by catalase. In addition, H2O2 decreased the ATP level and the [ATP]:[ADP] ratio measured in the presence of oligomycin reflecting an inhibition of glycolysis by H2O2, but this effect was not reversible. The effect of H2O2 on delta(psi)m in the presence of the complex I inhibitor, rotenone, was also unaltered by addition of catalase. These results provide circumstantial evidence for a relationship between the decreased NAD(P)H level and the inability of mitochondria to maintain delta(psi)m during oxidative stress.


Subject(s)
Mitochondria/physiology , NADP/metabolism , Oxidative Stress , Synaptosomes/metabolism , Animals , Glycolysis , Guinea Pigs , Hydrogen Peroxide/pharmacology , In Vitro Techniques , Membrane Potentials , Oligomycins/pharmacology , Oxidants/pharmacology
10.
J Neurosci ; 20(6): 2094-103, 2000 Mar 15.
Article in English | MEDLINE | ID: mdl-10704483

ABSTRACT

We have explored the consequences of a [Na(+)](i) load and oxidative stress in isolated nerve terminals. The Na(+) load was achieved by veratridine (5-40 microM), which allows Na(+) entry via a voltage-operated Na(+) channel, and oxidative stress was induced by hydrogen peroxide (0.1-0.5 mM). Remarkably, neither the [Na(+)](i) load nor exposure to H(2)O(2) had any major effect on [Ca(2+)](i), mitochondrial membrane potential (Deltapsim), or ATP level. However, the combination of an Na(+) load and oxidative stress caused ATP depletion, a collapse of Deltapsim, and a progressive deregulation of [Ca(2+)](i) and [Na(+)](i) homeostasis. The decrease in the ATP level was unrelated to an increase in [Ca(2+)](i) and paralleled the rise in [Na(+)](i). The loss of Deltapsim was prevented in the absence of Ca(2+) but unaltered in the presence of cyclosporin A. We conclude that the increased ATP consumption by the Na,K-ATPase that results from a modest [Na(+)](i) load places an additional demand on mitochondria metabolically compromised by an oxidative stress, which are unable to produce a sufficient amount of ATP to fuel the ATP-driven ion pumps. This results in a deregulation of [Na(+)](i) and [Ca(2+)](i), and as a result of the latter, collapse of Deltapsim. The vicious cycle generated in the combined presence of Na(+) load and oxidative stress could be an important factor in the neuronal injury produced by ischemia or excitotoxicity, in which the oxidative insult is superimposed on a disturbed Na(+) homeostasis.


Subject(s)
Adenosine Triphosphate/metabolism , Calcium/metabolism , Oxidative Stress/physiology , Presynaptic Terminals/enzymology , Sodium/metabolism , Animals , Calcium Channels/metabolism , Calcium-Transporting ATPases/antagonists & inhibitors , Calcium-Transporting ATPases/metabolism , Cell Membrane/enzymology , Cerebral Cortex , Dizocilpine Maleate/pharmacology , Electric Impedance , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Guinea Pigs , Homeostasis/physiology , Hydrogen Peroxide/pharmacology , Mitochondria/drug effects , Mitochondria/enzymology , Ouabain/pharmacology , Oxidants/pharmacology , Oxidative Stress/drug effects , Presynaptic Terminals/chemistry , Presynaptic Terminals/drug effects , Receptors, Glutamate/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Synaptosomes/chemistry , Synaptosomes/drug effects , Synaptosomes/enzymology , Tetrodotoxin/pharmacology , Veratridine/pharmacology
11.
J Neurosci ; 20(24): 8972-9, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11124972

ABSTRACT

In this study we addressed the function of the Krebs cycle to determine which enzyme(s) limits the availability of reduced nicotinamide adenine dinucleotide (NADH) for the respiratory chain under H(2)O(2)-induced oxidative stress, in intact isolated nerve terminals. The enzyme that was most vulnerable to inhibition by H(2)O(2) proved to be aconitase, being completely blocked at 50 microm H(2)O(2). alpha-Ketoglutarate dehydrogenase (alpha-KGDH) was also inhibited but only at higher H(2)O(2) concentrations (>/=100 microm), and only partial inactivation was achieved. The rotenone-induced increase in reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] fluorescence reflecting the amount of NADH available for the respiratory chain was also diminished by H(2)O(2), and the effect exerted at small concentrations (/=100 microm) inhibition of alpha-ketoglutarate dehydrogenase limits the amount of NADH available for the respiratory chain, and (4) increased consumption of NADPH makes a contribution to the H(2)O(2)-induced decrease in the amount of reduced pyridine nucleotides. These results emphasize the importance of alpha-KGDH in impaired mitochondrial function under oxidative stress, with implications for neurodegenerative diseases and cell damage induced by ischemia/reperfusion.


Subject(s)
Citric Acid Cycle/drug effects , Hydrogen Peroxide/pharmacology , Ketoglutarate Dehydrogenase Complex/metabolism , NAD/biosynthesis , Oxidative Stress/drug effects , Aconitate Hydratase/antagonists & inhibitors , Aconitate Hydratase/metabolism , Animals , Carmustine/pharmacology , Cerebral Cortex/chemistry , Cerebral Cortex/enzymology , Citrate (si)-Synthase/metabolism , Citrate (si)-Synthase/physiology , Dose-Response Relationship, Drug , Electron Transport/drug effects , Enzyme Activation/drug effects , Glucose/metabolism , Glutamic Acid/metabolism , Glutathione Reductase/antagonists & inhibitors , Glutathione Reductase/metabolism , Guinea Pigs , Ketoglutarate Dehydrogenase Complex/antagonists & inhibitors , Malate Dehydrogenase/metabolism , NADP/metabolism , Pyrimidines/metabolism , Rotenone/pharmacology , Spectrometry, Fluorescence , Succinate Dehydrogenase/metabolism , Synaptosomes/chemistry , Synaptosomes/enzymology
12.
J Neurochem ; 73(1): 220-8, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10386974

ABSTRACT

Mitochondrial membrane potential (delta psi(m)) was determined in intact isolated nerve terminals using the membrane potential-sensitive probe JC-1. Oxidative stress induced by H2O2 (0.1-1 mM) caused only a minor decrease in delta psi(m). When complex I of the respiratory chain was inhibited by rotenone (2 microM), delta psi(m) was unaltered, but on subsequent addition of H2O2, delta psi(m) started to decrease and collapsed during incubation with 0.5 mM H2O2 for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H2O2. H2O2 also induced a marked reduction in delta psi(m) when added after oligomycin (10 microM), an inhibitor of F0F1-ATPase. H2O2 (0.1 or 0.5 mM) inhibited alpha-ketoglutarate dehydrogenase and decreased the steady-state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine delta psi(m) in the presence of H2O2: (a) The NADH level reduced owing to inhibition of alpha-ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of delta psi(m) when the F0F1-ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H2O2 prevents maintenance of delta psi(m) by F0F1-ATPase. The results indicate that to maintain delta psi(m) in the nerve terminal during H2O2-induced oxidative stress, both complex I and F0F1-ATPase must be functional. Collapse of delta psi(m) could be a critical event in neuronal injury in ischemia or Parkinson's disease when H2O2 is generated in excess and complex I of the respiratory chain is simultaneously impaired.


Subject(s)
Hydrogen Peroxide/pharmacology , Ketoglutarate Dehydrogenase Complex/antagonists & inhibitors , Membrane Potentials , Mitochondria/ultrastructure , Nerve Endings/ultrastructure , Oxidative Stress/physiology , Animals , Benzimidazoles , Carbocyanines , Cerebral Cortex/ultrastructure , Enzyme Inhibitors/pharmacology , Fluorescent Dyes , Guinea Pigs , Intracellular Membranes/physiology , Membrane Potentials/drug effects , NADP/metabolism , Oligomycins/pharmacology , Oxidative Stress/drug effects , Proton-Translocating ATPases/antagonists & inhibitors , Rotenone/pharmacology , Spectrometry, Fluorescence , Synaptosomes/ultrastructure , Uncoupling Agents/pharmacology
14.
Neuroreport ; 9(8): 1849-53, 1998 Jun 01.
Article in English | MEDLINE | ID: mdl-9665614

ABSTRACT

The effect of the neuroprotective drug, vinpocetine on the veratridine-evoked [Na+]i and [Ca2+]i rise in isolated nerve terminals was studied. Vinpocetine, in a pharmacologically relevant concentration range (0.4-10 microM)i reduced the increase of [Na+]i induced by veratridine (100 microM). The effect of the drug was concentration-dependent with 10 microM vinpocetine completely preventing the increase of [Na+]i. The [Ca2+]i rise in response to veratridine was also prevented by vinpocetine. In addition, the [Ca2+]i signal induced by depolarization with 20 mM K+ was reduced by vinpocetine (1-20 microM). This effect was not influenced by preincubation with 1 microM TTX and was also observed when Na+ was replaced by N-methyl glucamine in the medium. It is concluded that vinpocetine is capable of inhibiting voltage-dependent Na+ and Ca2+ channels, respectively, and these effects might contribute to the neuroprotection exerted by the drug.


Subject(s)
Calcium/metabolism , Neuroprotective Agents/pharmacology , Sodium/metabolism , Synaptosomes/drug effects , Veratridine/pharmacology , Vinca Alkaloids/pharmacology , Animals , Guinea Pigs , In Vitro Techniques , Membrane Potentials/drug effects , Synaptosomes/metabolism
15.
Mol Pharmacol ; 53(4): 734-41, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9547365

ABSTRACT

The effect of the protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) was studied on the intracellular [Na+], pH, and plasma membrane potential in isolated nerve terminals. FCCP induced a rise of [Na+]i at, and even below, the concentrations (0.025-1 microM) in which it is usually used in intact cells to eliminate Ca2+ uptake by mitochondria. The FCCP-induced increase of [Na+]i correlates with a fall in both the ATP level and the ATP/ADP ratio. In addition, a sudden rise of the intracellular proton concentration ([H+]i) from 83 +/- 0.4 to 124 +/- 0.7 nM was observed on the addition of FCCP (1 microM). Parallel with the rise in [H+]i, an abrupt depolarization was detected, followed by a slower decrease in the plasma membrane potential. Both the extent of the pHi change and the fast depolarization of the plasma membrane were proportional to the proton electrochemical gradient across the plasma membrane; when this gradient was increased, greater depolarization was detected. The slower decrease of the membrane potential after the fast initial depolarization was abolished when the medium contained no Na+. It is concluded that FCCP (1) gives rise to a depolarization by setting the plasma membrane potential close to the proton equilibrium potential and (2) enhances the intracellular [Na+] as a consequence of an insufficient ATP level and ATP/ADP ratio to fuel the Na+,K+/ATPase. Because both disturbed Na+ homeostasis and plasma membrane depolarization could profoundly interfere with Ca2+ homeostasis in the presence of protonophores, consideration given to these alterations may help to clarify the cellular Ca2+ sequestration processes.


Subject(s)
Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Homeostasis/drug effects , Ionophores/pharmacology , Nerve Endings/drug effects , Sodium/physiology , Animals , Cell Membrane/drug effects , Cell Membrane/physiology , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Cerebral Cortex/physiology , Electrochemistry , Guinea Pigs , Hydrogen-Ion Concentration/drug effects , Intracellular Fluid/metabolism , Membrane Potentials/drug effects , Nerve Endings/metabolism , Nerve Endings/physiology , Sodium/metabolism , Synaptosomes/drug effects , Synaptosomes/metabolism , Synaptosomes/physiology
16.
Neurochem Int ; 33(6): 541-9, 1998 Dec.
Article in English | MEDLINE | ID: mdl-10098724

ABSTRACT

All-trans retinoic acid (RA), a potent inducer of neural development in non-committed neuroectodermal precursors and also, a teratogenic agent for early prosencephalic development is reported to promote the survival and differentiation of embryonic forebrain neurons, in vitro. In cultures of embryonic (E13, E15) rat forebrain cells, long-term (2-5 days) treatment with RA increased the number of neurons and the overall neurofilament immunoreactivity. Treatment with RA for periods longer than 1 h resulted in enhanced binding of the non-competitive NMDA-receptor antagonist, TCP, by embryonic and fetal (E17, E18) cells, but not by cells derived from perinatal (E19, P0) forebrains. As TCP binding-sites are localised within the channel-complex, treatment with RA was thought to result in an opening of the NMDA receptor channel. In direct binding assays, however, RA had no detectable effect, while conditioned media taken from RA-treated embryonic or fetal cells increased the TCP-binding, immediately. Analyses on conditioned media taken from control cultures of cells with various in vivo or in vitro ages revealed a stable extracellular glutamate level ([Glu]e) of 1-3 microM. This basal [Glu]e was restored within 24 h after addition of 100 microM exogenous glutamate. In the presence of RA, however, [Glu]e was stabilised at an approximately three-fold higher (4-10 microM) level by cells derived from embryonic and fetal brains. RA-treatment did not influence the [Glu]e in cultures of perinatal cells. The RA-induced rise in the neurofilament-immunoreactivity of embryonic brain cell cultures was prevented by simultaneous treatment with APV, a competitive antagonist of NMDA-receptors. The data suggest that a RA-induced shift in the set-point of extracellular glutamate-balance plays an important role in the promotion of survival and maturation of developing neurons, in culture.


Subject(s)
Prosencephalon/drug effects , Tretinoin/pharmacology , Animals , Extracellular Space/metabolism , Glutamic Acid/metabolism , Immunohistochemistry , Ion Channel Gating , N-Methylaspartate/pharmacology , Prosencephalon/cytology , Prosencephalon/embryology , Rats , Rats, Wistar , Receptors, N-Methyl-D-Aspartate/drug effects
17.
J Neurochem ; 69(6): 2529-37, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9375686

ABSTRACT

Oxidative insult elicited by hydrogen peroxide (H2O2) was previously shown to increase the basal intracellular Ca2+ concentration in synaptosomes. In the present study, the effect of H2O2 on the depolarization-evoked [Ca2+] signal was investigated. Pretreatment of synaptosomes with H2O2 (0.1-1 mM) augmented the [Ca2+] rise elicited by high K+ depolarization with essentially two alterations, the sudden sharp rise of [Ca2+]i due to K+ depolarization is enhanced and, instead of a decrease to a stable plateau, a slow, steady rise of [Ca2+]i follows the peak [Ca2+]i. H2O2 in the same concentration range lowered the ATP level and the [ATP]/[ADP] ratio. When carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) (1 microM) or rotenone (2 microM)/oligomycin (10 microM) was applied initially to block mitochondrial ATP production, the lowered [ATP]/[ADP] ratio was further reduced by subsequent addition of 0.5 mM H2O2. The decline of the [ATP]/[ADP] ratio was parallel with but could not explain the enhanced K+-evoked [Ca2+]i signal, indicated by experiments in which the [ATP]/[ADP] ratio was decreased by FCCP (0.1 microM) or rotenone (2 microM) to a similar value as by H2O2 without causing any alteration in the [Ca2+]i signal. These results indicate that H2O2-evoked oxidative stress, in its early phase, gives rise to a complex dysfunction in the Ca2+ homeostasis and, parallel with it, to an impaired energy status.


Subject(s)
Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Calcium/physiology , Oxidative Stress/physiology , Signal Transduction/physiology , Synaptosomes/physiology , Animals , Calcium/metabolism , Electrophysiology , Energy Metabolism/drug effects , Guinea Pigs , Hydrogen Peroxide/pharmacology , Intracellular Membranes/metabolism , Osmolar Concentration , Oxidants/pharmacology , Potassium/pharmacology , Synaptosomes/metabolism
18.
Stroke ; 28(5): 988-92, 1997 May.
Article in English | MEDLINE | ID: mdl-9158639

ABSTRACT

BACKGROUND AND PURPOSE: Coronary artery disease is prevalent in stroke patients and is an important factor affecting rehabilitation and health outcomes. However, the presence of neurological deficits in gait and balance has discouraged systematic application of exercise testing and prescription in the stroke population. We evaluated a novel graded treadmill stress test in paretic stroke patients and tested floor walking as a predictor of adequate neurological function to perform the treadmill test. METHODS: Patients (n = 31) with residual paretic gait deficits after ischemic stroke were evaluated with graded treadmill at gait velocities individualized to functional mobility observed during an initial zero-incline treadmill tolerance test. RESULTS: Most patients (30/31) tolerated testing, achieving mean heart rates of 129 +/- 14 beats per minute (mean +/- SD), representing 84 +/- 10% of maximal age-predicted heart rate. Evidence for asymptomatic myocardial ischemia was found in 29% of those without known coronary artery disease. Exercise termination was more often due to generalized fatigue than cardiopulmonary intolerance (23/31 versus 4/31; P < .0001) or hemiparetic leg fatigue (1/31; P < .0001). Floor walking across a wide range of velocities (0.25 to 2.5 mph) demonstrated a strong linear relation with treadmill velocities (n = 24; r = 80; P < .0001); all patients floor walking at > or = 0.5 mph had adequate neuromotor function to perform the exercise test. CONCLUSIONS: These findings suggest that graded treadmill exercise testing, with proper safety precautions, can be used to assess cardiopulmonary function in paretic stroke patients. A simple floor-walking test predicts adequate neurological function to perform the exercise test. Exercise capacity is most limited by generalized fatigue and not by the paretic limb, supporting a rationale for endurance training in this population.


Subject(s)
Cerebrovascular Disorders/diagnosis , Exercise Test , Hemiplegia/diagnosis , Aged , Cerebrovascular Disorders/physiopathology , Female , Gait , Hemiplegia/physiopathology , Humans , Male , Middle Aged , Physical Endurance
19.
Stroke ; 28(2): 326-30, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9040684

ABSTRACT

BACKGROUND AND PURPOSE: Elevated energy costs of hemiparetic gait contribute to functional disability after stroke, particularly in physically deconditioned older patients. We investigated the effects of 6 months of treadmill aerobic exercise training on the energy expenditure and cardiovascular demands of submaximal effort ambulation in stroke patients with chronic hemiparetic gait. METHODS: Nine older stroke patients with chronic hemiparetic gait were enrolled in a 6-month program of low-intensity aerobic exercise using a graded treadmill. Repeated measures of energy expenditure based on steady state oxygen consumption during a standardized 1-mph submaximal effort treadmill walking task were performed before and after training. RESULTS: Six months of exercise training produced significant reductions in energy expenditure (n = 9; 3.40 +/- 0.27 versus 2.72 +/- 0.25 kcal/min [mean +/- SEM]; P < .005) during a given submaximal effort treadmill walking task. Repeated measures analysis in the subset of patients (n = 8) tested at baseline and after 3 and 6 months revealed that reductions in energy expenditure were progressive (F = 11.1; P < .02) and that exercise-mediated declines in both oxygen consumption (F = 9.7; P < .02) and respiratory exchange ratio (F = 13.4; P < .01) occurred in a strong linear pattern. These stroke patients could perform the same standardized submaximal exercise task at progressively lower heart rates after 3 months (96 +/- 4 versus 87 +/- 4 beats per minute) and 6 months of training (82 +/- 4 beats per minute; F = 35.4; P < .002). CONCLUSIONS: Six months of low-intensity treadmill endurance training produces substantial and progressive reductions in the energy expenditure and cardiovascular demands of walking in older patients with chronic hemiparetic stroke. This suggests that task-oriented aerobic exercise may improve functional mobility and the cardiovascular fitness profile in this population.


Subject(s)
Brain Ischemia/rehabilitation , Exercise Therapy , Exercise , Gait , Hemiplegia/rehabilitation , Movement Disorders/physiopathology , Aged , Brain Ischemia/complications , Cardiovascular Diseases/complications , Cardiovascular Diseases/physiopathology , Diabetes Complications , Energy Metabolism , Exercise Therapy/instrumentation , Hemiplegia/etiology , Hemodynamics , Humans , Hyperlipidemias/complications , Male , Middle Aged , Movement Disorders/etiology , Smoking , Walking
20.
J Neurochem ; 66(5): 2057-66, 1996 May.
Article in English | MEDLINE | ID: mdl-8780036

ABSTRACT

The effects of peroxides were investigated on the membrane potential, intracellular Na+ ([Na+]i) and intracellular Ca2+ ([Ca2+]i) concentrations, and basal glutamate release of synaptosomes. Both H2O2 and the organic cumene hydroperoxide produced a slow and continuous depolarization, parallel to an increase of [Na+]i over an incubation period of 15 min. A steady rise of the [Ca2+]i due to peroxides was also observed that was external Ca2+ dependent and detected only at an inwardly directed Ca2+ gradient of the plasma membrane. These changes did not correlate with lipid peroxidation, which was elicited by cumene hydroperoxide but not by H2O2. Resting release of glutamate remained unchanged during the first 15 min of incubation in the presence of peroxides. These alterations may indicate early dysfunctions in the sequence of events occurring in the nerve terminals in response to oxidative stress.


Subject(s)
Calcium/metabolism , Intracellular Membranes/metabolism , Nerve Endings/drug effects , Peroxides/pharmacology , Reactive Oxygen Species/physiology , Sodium/metabolism , Animals , Benzene Derivatives/pharmacology , Electrophysiology , Glutamic Acid/metabolism , Guinea Pigs , Hydrogen Peroxide/pharmacology , Lipid Peroxides/metabolism , Membrane Potentials/drug effects , Osmolar Concentration , Synaptosomes/drug effects , Synaptosomes/physiology
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