IN VITRO STUDIES ON THE INTERACTION OF BRAIN MONOAMINE OXIDASE WITH 5,6- AND 5,7-DIHYDROXYTRYPTAMINE12

[¹⁴C]5,6-Dihydroxytryptamine ([¹⁴C] 5,6-DHT) and [¹⁴C]5,7-dihydroxytryptamine ([¹⁴C]5,7-DHT) were deaminated to toluene-isoamylalcohol extractable products when incubated with homogenates of rat hypothalamus or pons-medulla oblongata. [¹⁴C]5,6-Dihydroxyindole acetic acid ([¹⁴C]5.6-DHIAA) and [¹⁴C]5,7-dihydroxyindole acetic acid ([¹⁴C]5,7-DHIAA) were detected as MAO metabolites by TLC besides non-identified components. The conversion of [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT obeyed, at least initially, Michaelis-Menten kinetics (K_m 5,7-DHT: 0.5 × 10^?3M; K_m 5,6-DHT: 1.25 × 10^?3M). Inhibition of the reaction by the MAO A inhibitor, clorgyline, resulted in a typical double sigmoidal inhibition curve indicating that both amines are metabolized by both types of MAO (A and B). In deprenyl inhibition studies, however, 5,7- and 5,6-DHT seemed to be preferred substrates of MAO A. Incubation of rat brain homogenates with [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT or with the MAO metabolites [¹⁴C]5,6-DHIAA and [¹⁴C]5,7-DHIAA caused a time-dependent break-down of the dihydroxylated indole compounds with subsequent binding of radioactivity to perchloric acid insoluble tissue components. 5,6-DHT inactivated MAO in rat brain homogenates parallel to its decomposition and extensive protein binding. The inactivation of MAO by 5,6-DHT and the extensive binding of radioactivity to protein were antagonized by dithiothreitol (DTT), glutathione (GSH) and L-ascorbic acid. Reduction of [O₂] in the incubation medium slightly attenuated the inactivation of MAO by 5,6-DHT. Catalase or superoxide dismutase failed to prevent MAO from being inactivated by 5,6-DHT. The results suggest that oxidation products of 5,6-DHT, e.g. its corresponding o-quinone, are involved in the inactivation of MAO in vitro and mainly responsible for the binding of radioactivity to brain proteins in vitro. Similar mechanisms may also be operative in the in vivo neurotoxicity of 5,6-DHT. The lack of inactivation of MAO by 5,7-DHT in vitro correlated with a low degree of radioactivity binding (from [¹⁴C]5,7-DHT) to homogenate protein pellets; the binding to proteins was barely influenced by GSH, cysteine, DTT and l -ascorbic acid. These latter findings do not provide a plausible explanation for the mechanism(s) involved in the well known in vivo neurotoxicity of 5,7-DHT.     

Peroxidase-promoted oxidation and peroxidation of the serotonergic neurotoxin 5,7-dihydroxytryptamine

Spectral data provide the first evidence that lactoperoxidase, a model enzyme for most mammalian peroxidases, catalyzed the one-electron oxidation and/or peroxidation of 5,7-dihydroxytryptamine. This process correlates with the production of superoxide radicals as is evident from the observed inhibitory effect of superoxide dismutase on product formation. 5,7-Dihydroxytryptamine is a classical peroxidase-oxidase substrate acting as a one-electron donor for enzyme compounds I, II and III. The one-electron peroxidatic oxidation of this serotonergic neurotoxin, responsible for the selective degeneration of central (5-hydroxytryptamine) neurons, is a fast process requiring measurement on the ms time scale. Attention is drawn to the biochemical and toxicological implications, because this fast reaction results in formation of known cell damaging species: free radicals, superoxide radicals and quinoidal products probably involved in the toxic action of 5,7-dihydroxytryptamine.Abbreviations 5-HT 5-Hydroxytryptamine, Serotonin - 5,7-DHT 5,7-Dihydroxytryptamine - 5,6-DHT 5,6-Dihydroxytryptamine - 5-HT-4,7-Dione 5-Hydroxytryptamine-4,7-Dione - GPO Glutathione Peroxidase - MAO Monoamine Oxidase - LPO Lactoperoxidase, the Roman numerals I, II and III added to LPO indicate compounds I, III and III of the enzyme - TPO Thyroid Peroxidase - IPO Intestinal Peroxidase - UPO Uterine Peroxidase - EPO Eosinophil Peroxidase - SOD Superoxide Dismutase - DPPH 1,1-Diphenyl-2-Picrylhydrazil radical - _max absorption maxima - ESR Electron Spin Resonance     

The simultaneous determination of carbon dioxide release and oxygen uptake in suspensions of plant leaf mitochondria oxidizing glycine

The construction and operation of a device for continuous measurement of CO₂ release by suspensions of respiring mitochondria is described. A combination of this device with a Clark-type O₂ electrode was used for simultaneous measurement of respiration and of CO₂ release by spinach and pea leaf mitochondria with glycine as substrate. Both mitochondrial preparations showed high rates of respiration and high respiratory control ratios. The addition of oxaloacetate not only inhibited O₂ uptake substantially, but also greatly stimulated glycine oxidation as monitored by CO₂ release. In spinach leaf mitochondria, the maximal rates of glycine oxidation thus obtained, were two times higher than the rate of glycine oxidation required at average rates of photorespiration. It is concluded from these results that under saturating conditions the capacity of glycine oxidation by intact mitochondria exceeds the capacity of glycine-dependent respiration.     

Hydroxyl Radical-Mediated Oxidation of Serotonin: Potential Insights into the Neurotoxicity of Methamphetamine

Abstract: When incubated with a hydroxyl radical (HO^?)-generating system (ascorbic acid/Fe²⁺-EDTA/O₂/H₂O₂), 5-hydroxytryptamine (5-HT; serotonin) is rapidly oxidized initially to a mixture of 2,5-, 4,5-, and 5,6-dihydroxytryptamine (DHT). The major reaction product is 2,5-DHT, which at physiological pH exists as its keto tautomer, 5-hydroxy-3-ethylamino-2-oxindole (5-HEO). Rapid autoxidation of 4,5-DHT gives tryptamine-4,5-dione (T-4,5-D), which reacts with the C(3)-centered carbanion of 5-HEO to give 3,3′-bis(2-aminoethyl)-5-hydroxy-[3,7′-bi-1H-indole]-2,4′,5′-3H-trione (7). The latter slowly cyclizes to 3′-(2-aminoethyl)-1′,6′,7′,8′-tetrahydro-5-hydroxyspiro[3H-indole-3,9′-[9H]pyrrolo[2,3-f]quinoline]-2,4′,5′(1H)- trione (9). A minor amount of T-4,5-D dimerizes to give 7,7′-bi-(5-hydroxytryptamine-4-one) (7,7′-D). In the presence of GSH, the reaction of T-4,5-D with 5-HEO is diverted and, in the presence of sufficient concentrations of this tripeptide, completely blocked. This is because GSH preferentially reacts with T-4,5-D to give 7-S-glutathionyltryptamine-4,5-dione (11). The results of this investigation suggest that 5,6-DHT, 5-HEO, 7, and 9 are products unique to the HO^?-mediated oxidation of 5-HT. Thus, the observation of other investigators that 5,6-DHT is formed in the brains of rats following a large dose of methamphetamine (MA) suggests that this drug might evoke HO^? formation. However, the present in vitro study indicates that 5,6-DHT is a rather minor, unstable product of the HO^?-mediated oxidation of 5-HT and suggests that detection of 5-HEO, 7/9, and 11 in rat brain following MA administration could provide additional support for HO^? formation. Furthermore, one or more of the intermediates and major products of oxidation of 5-HT by HO^? might, in addition to 5,6-DHT, contribute to the MA-induced degeneration of serotonergic neurons.     

Molecular mechanism of biological action of the serotonergic neurotoxin 5,7-dihydroxytryptamine

5,7-Dihydroxytryptamine (5,7-DHT) is a selective serotonergic neurotoxin by virtue of its selective uptake into 5-hydroxytryptamine neurons and its ability to undergo autoxidation. The mechanism by which 5,7-DHT induces neurodegenerative effects remains enigmatic. The mechanism of autoxidation of 5,7-DHT, which has been recently discovered, is unique among the autoxidizable neurotoxins and involves incorporation of oxygen to produce the 4-hydroperoxy-5-keto derivative of 5,7-DHT and thence the (4,7) p-quinone of 4,5,7-trihydroxytryptamine (4,5,7-THTQ), a relatively unreactive quinone. In addition, no reduced oxygen species such as hydrogen peroxide, superoxide and hydroxyl radical are produced during autoxidation of 5,7-DHT. Yet, there is evidence to suggest that both the covalent modification of endogenous macromolecules by 5,7-DHT derived products and the toxic effects of reduced oxygen species are, at least in part, responsible for the neurodegenerative effects of 5,7-DHT. Here we propose that (1) the 4-hydroperoxy-5-keto derivative of 5,7-DHT may serve as a substrate for glutathione peroxidase to eventually produce reduced oxygen species and 4,5,7-THTQ, (2) 4,5,7-THTQ may undergo redox cycling thereby generating reduced oxygen species and lowering the reducing equivalents of the neuron, (3) rapid oxygen consumption by 5,7-DHT and the products derived from it may lead to hypoxia, and (4) the product of autoxidation of 5,7-dihydroxyindole-3-acetaldehyde, the monoamine oxidase metabolite of 5,7-DHT, may serve as an alkylating (crosslinking) agent of proteins.     

Respiration in Adipocytes is Inhibited by Reactive Oxygen Species

It is a desirable goal to stimulate fuel oxidation in adipocytes and shift the balance toward less fuel storage and more burning. To understand this regulatory process, respiration was measured in primary rat adipocytes, mitochondria, and fat‐fed mice. Maximum O₂ consumption, in vitro, was determined with a chemical uncoupler of oxidative phosphorylation (carbonylcyanide p‐trifluoromethoxyphenylhydrazone (FCCP)). The adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio was measured by luminescence. Mitochondria were localized by confocal microscopy with MitoTracker Green and their membrane potential (Δψ_M) measured using tetramethylrhodamine ethyl ester perchlorate (TMRE). The effect of N‐acetylcysteine (NAC) on respiration and body composition in vivo was assessed in mice. Addition of FCCP collapsed Δψ_M and decreased the ATP/ADP ratio. However, we demonstrated the same rate of adipocyte O₂ consumption in the absence or presence of fuels and FCCP. Respiration was only stimulated when reactive oxygen species (ROS) were scavenged by pyruvate or NAC: other fuels or fuel combinations had little effect. Importantly, the ROS scavenging role of pyruvate was not affected by rotenone, an inhibitor of mitochondrial complex I. In addition, mice that consumed NAC exhibited increased O₂ consumption and decreased body fat in vivo. These studies suggest for the first time that adipocyte O₂ consumption may be inhibited by ROS, because pyruvate and NAC stimulated respiration. ROS inhibition of O₂ consumption may explain the difficulty to identify effective strategies to increase fat burning in adipocytes. Stimulating fuel oxidation in adipocytes by decreasing ROS may provide a novel means to shift the balance from fuel storage to fuel burning.     

Nicotinamide Nucleotide Transhydrogenase (Nnt) Links the Substrate Requirement in Brain Mitochondria for Hydrogen Peroxide Removal to the Thioredoxin/Peroxiredoxin (Trx/Prx) System

Mitochondrial reactive oxygen species are implicated in the etiology of multiple neurodegenerative diseases, including Parkinson disease. Mitochondria are known to be net producers of ROS, but recently we have shown that brain mitochondria can consume mitochondrial hydrogen peroxide (H₂O₂) in a respiration-dependent manner predominantly by the thioredoxin/peroxiredoxin system. Here, we sought to determine the mechanism linking mitochondrial respiration with H₂O₂ catabolism in brain mitochondria and dopaminergic cells. We hypothesized that nicotinamide nucleotide transhydrogenase (Nnt), which utilizes the proton gradient to generate NADPH from NADH and NADP⁺, provides the link between mitochondrial respiration and H₂O₂ detoxification through the thioredoxin/peroxiredoxin system. Pharmacological inhibition of Nnt in isolated brain mitochondria significantly decreased their ability to consume H₂O₂ in the presence, but not absence, of respiration substrates. Nnt inhibition in liver mitochondria, which do not require substrates to detoxify H₂O₂, had no effect. Pharmacological inhibition or lentiviral knockdown of Nnt in N27 dopaminergic cells (a) decreased H₂O₂ catabolism, (b) decreased NADPH and increased NADP⁺ levels, and (c) decreased basal, spare, and maximal mitochondrial oxygen consumption rates. Nnt-deficient cells possessed higher levels of oxidized mitochondrial Prx, which rendered them more susceptible to steady-state increases in H₂O₂ and cell death following exposure to subtoxic levels of paraquat. These data implicate Nnt as the critical link between the metabolic and H₂O₂ antioxidant function in brain mitochondria and suggests Nnt as a potential therapeutic target to improve the redox balance in conditions of oxidative stress associated with neurodegenerative diseases.     

Oxygen consumption by purified plasmalemma vesicles from wheat roots: Stimulation by NADH and salicylhydroxamic acid (SHAM)

Plasmalemma vesicles from wheat (Triticum aestivum L.) roots consumed O₂ and the addition of 1 mM NADH increased the rate ~ 3-fold (to 15-30 nmol O₂·mg⁻¹·min⁻¹). The NADH-dependent O₂ uptake was abolished by catalase. In the presence of salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase pathway in plant mitochondria, NADH-dependent O₂ consumption was stimulated 10–20-fold (to 200–400 nmol·mg^1̄·min⁻¹). Catalase also abolished this stimulation, which was KCN-sensitive but antimycin A-insensitive, and the production of H₂O₂ during SHAM-stimulated NADH-dependent O₂ uptake was demonstrated. Irrespective of the mechanism, SHAM-stimulated respiration by root plasmalemma makes it difficult to interpret results on root respiration obtained using KCN and SHAM.     

Respiration-dependent H2O2 Removal in Brain Mitochondria via the Thioredoxin/Peroxiredoxin System

Mitochondrial reactive oxygen species (ROS) play an important role in both physiological cell signaling processes and numerous pathological states, including neurodegenerative disorders such as Parkinson disease. While mitochondria are considered the major cellular source of ROS, their role in ROS removal remains largely unknown. Using polarographic methods for real-time detection of steady-state H₂O₂ levels, we were able to quantitatively measure the contributions of potential systems toward H₂O₂ removal by brain mitochondria. Isolated rat brain mitochondria showed significant rates of exogenous H₂O₂ removal (9–12 nmol/min/mg of protein) in the presence of substrates, indicating a respiration-dependent process. Glutathione systems showed only minimal contributions: 25% decrease with glutathione reductase inhibition and no effect by glutathione peroxidase inhibition. In contrast, inhibitors of thioredoxin reductase, including auranofin and 1-chloro-2,4-dinitrobenzene, attenuated H₂O₂ removal rates in mitochondria by 80%. Furthermore, a 50% decrease in H₂O₂ removal was observed following oxidation of peroxiredoxin. Differential oxidation of glutathione or thioredoxin proteins by copper (II) or arsenite, respectively, provided further support for the thioredoxin/peroxiredoxin system as the major contributor to mitochondrial H₂O₂ removal. Inhibition of the thioredoxin system exacerbated mitochondrial H₂O₂ production by the redox cycling agent, paraquat. Additionally, decreases in H₂O₂ removal were observed in intact dopaminergic neurons with thioredoxin reductase inhibition, implicating this mechanism in whole cell systems. Therefore, in addition to their recognized role in ROS production, mitochondria also remove ROS. These findings implicate respiration- and thioredoxin-dependent ROS removal as a potentially important mitochondrial function that may contribute to physiological and pathological processes in the brain.     

Direct evidence for the presence of a rotenone-resistant NADH dehydrogenase on the inner surface of the inner membrane of plant mitochondria

Submitochondrial particles (SMP) were produced from Jerusalem artichoke (Helianthus tuberosus L.) mitochondria by sonication and differential centrifugation. The SMP were about 50% inside-out as measured by the access of reduced cytochrome c to cytochrome c oxidase. Uncoupled NADH oxidation (1 mM NADH) by the SMP was 120 nmol O₂ min^?1mg^?1, which was reduced to 98 nmol O₂ min^?1 (mg mitochondrial protein)^?1 in the presence of EGTA. In contrast, the oxidation of NADH by intact mitochondria was completely inhibited by EGTA (from 182 to 14 nmol O₂ min^?1mg^?1). The EGTA-resistant NADH oxidation by the SMP is ascribed to the NADH dehydrogenase(s) on the inside of the inner membrane and exposed to the medium in the inside-out SMP. In the presence of EGTA it could be shown that two NADH dehydrogenase activities were present in the SMP. One had an apparent K_m of 7 μM for NADH, a V_max of 80 nmol NADH min^?1mg^?1, and was rotenone-sensitive. This dehydrogenase is equivalent to the mammalian Complex I NADH dehydrogenase. The other dehydrogenase, which was rotenone-resistant, had a K_m of 80 μM and a V_max of 131 nmol NADH min^?1mg^?1; it is probably responsible for the rotenone-resistant oxidation of organic acids often observed in plant mitochondria. The redox poise of the pyridine nucleotides had only a small effect on the relative rates of the two internal dehydrogenases. Electron flow through these dehydrogenases appears, therefore, to be regulated mainly by the concentration of NADH in the matrix of the mitochondria.     

Effects of equisetin on rat liver mitochondria: Evidence for inhibition of substrate anion carriers of the inner membrane

The effect of equisetin, an antibiotic produced byFusarium equiseti, has been studied on mitochondrial functions (respiration, ATPase, ion transport). Equisetin inhibits the DNP-stimulated ATPase activity of rat liver mitochondria and mitoplasts in a concentration-dependent manner; 50% inhibition is caused by about 8 nmol equisetin/mg protein. The antibiotic is without effect either on the ATPase activity of submitochondrial particles or on the purified F₁-ATPase. It inhibits both the ADP- or DNP-activated oxygen uptake by mitochondria in the presence of glutamate + malate or succinate as substrates, but only the ADP-stimulated respiration is inhibited if the electron donors are TMPD + ascorbate. It does not affect the NADH or succinate oxidation of submitochondrial particles. Equisetin inhibits in a concentration-dependent manner the active Ca²⁺-uptake of mitochondria energized both by ATP or succinate without affecting the Ca²⁺-uniporter itself. The antibiotic inhibits the ATP-uptake by mitochondria (50% inhibition at about 8 nmol equisetin/mg protein) and the P_i and dicarboxylate carrier. It does not lower the membrane potential at least up to 200 nmol/mg protein concentration. The data presented in this paper indicate that equisetin specifically inhibits the substrate anion carriers of the mitochondrial inner membrane.Abbreviations EGTA ethyleneglycol bis/-aminoethylether/-N, N-tetraacetic acid - DNP 2, 4-dinitrophenol - TMPD N,N,N,N,tetramethyl-p-phenylenediamine - CCP carbonylcyanide-m-chlorophenyl hydrazone - TPP tetraphenyl-phosphonium - Hepes /4,(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid/     

Unique features of flight muscles mitochondria of honey bees (Apis mellifera L.)

Honey bees Apis mellifera L. are one of the most studied insect species due to their economic importance. The interest in studying honey bees chiefly stems from the recent rapid decrease in their world population, which has become a problem of food security. Nevertheless, there are no systemic studies on the properties of the mitochondria of honey bee flight muscles. We conducted a research of the mitochondria of the flight muscles of A. mellifera L. The influence of various organic substrates on mitochondrial respiration in the presence or absence of adenosine diphosphate (ADP) was investigated. We demonstrated that pyruvate is the optimal substrate for the coupled respiration. A combination of pyruvate and glutamate is required for the maximal respiration rate. We also show that succinate oxidation does not support the oxidative phosphorylation and the generation of membrane potential. We also studied the production of reactive oxygen species by isolated mitochondria. The greatest production of H₂O₂ (as a percentage of the rate of oxygen consumed) in the absence of ADP was observed during the respiration supported by α‐glycerophosphate, malate, and a combination of malate with another NAD‐linked substrate. We showed that honey bee flight muscle mitochondria are unable to uptake Ca²⁺‐ions. We also show that bee mitochondria are able to oxidize the respiration substrates effectively at the temperature of 50°С compared to Bombus terrestris mitochondria, which were more adapted to lower temperatures.     

Growth and Mitochondrial Respiration of Mungbeans (Phaseolus aureus Roxb.) Germinated at Low Pressure

Mungbean (Phaseolus aureus Roxb.) seedlings were grown hypobarically to assess the effects of low pressure (21-24 kilopascals) on growth and mitochondrial respiration. Control seedlings grown at ambient pressure (101 kilopascals) were provided amounts of O₂ equivalent to those provided experimental seedlings at reduced pressure to factor out responses to O₂ concentration and to total pressure. Respiration was assayed using washed mitochondria, and was found to respond only to O₂ concentration. Regardless of total pressure, seedlings grown at 2 millimoles O₂ per liter had higher state 3 respiration rates and decreased percentages of alternative respiration compared to ambient (8.4 millimoles O₂ per liter) controls. In contrast, seedling growth responded to total pressure but not to O₂ concentration. Seedlings were significantly larger when grown under low pressure. While low O₂ (2 millimoles O₂ per liter) diminished growth at ambient pressure, growth at low pressure in the same oxygen concentration was enhanced. Respiratory development and growth of mungbean seedlings under low pressure is unimpaired whether oxygen or air is used as the chamber gas, and further, low pressure can improve growth under conditions of poor aeration.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in continuous cultures

The oxidation and growth kinetics of ferrous iron with Thiobacillus ferrooxidans in continuous cultures was examined at several total iron concentrations. On-line off-gas analyses of O₂ and CO₂ were used to measure the oxygen and carbon dioxide consumption rates in the culture. Off-line respiration measurements in a biological oxygen monitor (BOM) were used to measure directly the maximum specific oxygen consumption rate, qO₂,max, of cells grown in continuous culture. It was shown that these reproducibly measured values of qO₂,max vary with the dilution rate. The biomass-specific oxygen consumption rate, qO₂, is dependent on the ratio of the ferric and ferrous iron concentrations in the culture. The oxidation kinetics was accurately described with a rate equation for competitive ferric iron inhibition, using the value of qO₂,max measured in the BOM. Accordingly, only the kinetic constant Ks/K _i needed to be fitted from the measurements. A new method was introduced to determine the steady-state kinetics of a cell suspension in a batch culture that only takes a few hours. The batch culture was set up by terminating the feeding of a continuous culture at its steady state. The kinetic constant K _s/K _i determined in this batch culture agreed with the value determined in continuous cultures at various steady states. Received: 8 February 1999 / Accepted: 17 February 1999     

Evidence for H2O2 as the product of reduction of oxygen by alternative oxidase in mitochondria from potato tubers

Oxygen consumption by alternative oxidase (AOX), present in mitochondria of many angiosperms, is known to be cyanide-resistant in contrast to cytochrome oxidase. Its activity in potato tuber (Solanum tuberosum L.) was induced following chilling treatment at 4 °C. About half of the total O₂ consumption of succinate oxidation in such mitochondria was found to be sensitive to SHAM, a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive oxygen consumption by nearly half, and addition at the end of the reaction released nearly half of the consumed oxygen by AOX, both typical of catalase action on H₂O₂. These findings with catalase suggest that the product of reduction of AOX is H₂O₂ and not H₂O, as previously surmised. In potatoes subjected to chill stress (4 °C) for periods of 3, 5 and ?8 days the activity of AOX in mitochondria increased progressively with a corresponding increase in the AOX protein detected by immunoblot of the protein.     

Xylem sap flow as a major pathway for oxygen supply to the sapwood of birch (Betula pubescens Ehr.)

The role of xylem sap flow as an aqueous pathway for oxygen supply to the wood parenchyma of Betula pubescens saplings was investigated. Using micro‐optode sensors the oxygen status of the sapwood was quantified in relation to mass flow of xylem sap. Sap flow was gradually reduced by an increasing oxygen depletion in the root space. The effect of sap flow on radial O₂ transport between stem and atmosphere was assessed by a stoichiometrical approach between respiratory CO₂ production and O₂ consumption. Restriction of sap flow set in 36.5 h after the onset of O₂ depletion, and was complete after 71 h. Interruption of sap flow drastically increased the O₂ deficit in the sapwood to 70%. Sap flow contributed about 60% to the total oxygen supply to the sapwood. Diurnal O₂ flow rates varied between 3 and 6.3 nmol O₂ m^?2 leaf area (LA) s^?1 during night‐ and daytime, respectively. Maximum O₂ flow rates of 20 nmol O₂ m^?2 LA s^?1 were reached at highest sap flow rates of 5.7 mmol H₂O m^?2 LA s^?1. Sap flow not only affected the oxygen status of the sapwood but also had an effect on radial O₂ transport between stem and atmosphere.     

Oxidative phosphorylation of liver mitochondria from mice acclimatized to hypobaric hypoxia

Mice exposed to intermittent hypobaric hypoxia for 20 hours a day, 6 days a week, develop extracellular adaptive responses similar to those found in humans exposed to oxygen tension equivalent to that found at an altitude of 4500 m. Isolated liver mitochondria from these animals show no significant differences in rates of substrate-stimulated respiration, ADP-stimulated respiration and the respiratory control ratio (RCR), when compared with sea level controls. Undetectable or negligible differences in these parameters are also noted when sea level animals are exposed for one hour to severe hypoxia (7% O₂). We therefore conclude that the oxidative phosphorylation capacity of the isolated mouse liver mitochondria remains unaltered in both acute and chronic hypoxia. However thein vivo oxygen consumption by mice at this degree of hypoxia was markedly reduced. Lack of observable changes in oxidative phosphorylation could be accounted for by extracellular adaptations in mitochondria isolated from acclimatized animals. This explanation, however, is not consistent with the lack of changes on oxidative phosphorylation in mitochondria isolated from mice undergoing acute hypoxia at sea level. It is then suggested that isolated mitochondrial preparations are of limited value for investigating biochemical mechanisms underlying the variation of cellular respiration occurringin vivo.     

The quantitative measurement of H2O2 generation in isolated mitochondria

Adequate methods to measure the rate of mitochondrial oxygen radical generation are needed since oxygen radicals are involved in many pathologies. A fluorometric method appropriate to measure the rate of generation of H₂O₂ in intact mitochondria is described. Just after isolation of functional mitochondria from fresh tissues, rates of generation of H₂O₂ are kinetically measured by fluorometry in the presence of homovanillic acid and horseradish peroxidase. The method is specific for H₂O₂ and is sensitive enough to assay mitochondrial H₂O₂ generation in the presence of respiratory substrate without inhibitors of the respiratory chain. Simultaneous measurement of mitochondrial oxygen consumption allows calculation of the free radical leak: the percentage of electrons out of sequence which reduce oxygen to oxygen radicals along the mitochondrial respiratory chain instead of reducing oxygen to water at the terminal cytochrome oxidase. The method shows instantaneous response to H₂O₂. This makes it appropriate to study the quick effects of different inhibitors and modulators on the rate of mitochondrial oxygen radical production. Its application to the localization of the sites where caloric restriction decreases mitochondrial oxygen radical generation in heart mitochondria is described.     

O2 consumption and superoxide dismutase content in purified mitochondria from soybean root nodules

Soybean nodule mitochondria have been separated from cotnaminating organella on discontinuous Percoll gradients. The preparations appeared highly purified and at least 80% of the mitochondria were estimated to be derived from infected cells. Percoll-purified mitochondria showed important respiratory activity; in the case of succinate, the rate of O₂ consumption was 185 nmol O₂ min⁻¹ mg⁻¹ and the respiratory control and ADP/O ratio reached 2.72 and 1.16, respectively. These organelles also exhibited an active manganese containing superoxide dismutase (9.7 U mg⁻¹), whose purification is reported. These results are consistent with a significant O₂ consumption by host cell mitochondria in vivo and the possibility of a competition for O₂ supply with the bacteroids is discussed.     

Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle

Acute effects of free fatty acids (FFA) were investigated on: (1) glucose oxidation, and UCP-2 and -3 mRNA and protein levels in 1 h incubated rat soleus and extensor digitorium longus (EDL) muscles, (2) mitochondrial membrane potential in cultured skeletal muscle cells, (3) respiratory activity and transmembrane electrical potential in mitochondria isolated from rat skeletal muscle, and (4) oxygen consumption by anesthetized rats. Long-chain FFA increased both basal and insulin-stimulated glucose oxidation in incubated rat soleus and EDL muscles and reduced mitochondrial membrane potential in C2C12 myotubes and rat skeletal muscle cells. Caprylic, palmitic, oleic, and linoleic acid increased O₂ consumption and decreased electrical membrane potential in isolated mitochondria from rat skeletal muscles. FFA did not alter UCP-2 and -3 mRNA and protein levels in rat soleus and EDL muscles. Palmitic acid increased oxygen consumption by anesthetized rats. These results suggest that long-chain FFA acutely lead to mitochondrial uncoupling in skeletal muscle.