Respiratory activity of isolated hepatocytes

Oligomycin and uncoupler of oxidative phosphorylation have been studied for their effect on the respiration activity of hepatocytes in rats. The respiration rate in the presence of oligomycin and uncoupler is higher than it is with the respiration uncoupled in the absence of oligomycin. Exogenic succinate makes endogenic respiration of hepatocytes in the presence of digitonin 5 times more intensive. The obtained results evidence for the fact that the uncoupled respiration is limited by the concentration of substrates able to be oxidized in the respiration chain of mitochondria. Oligomycin induces accumulation of substrates and following addition of the disconnector evokes their fast oxidation.     

Inhibition of mitochondrial respiration by phosphoenolpyruvate

The cytosolic factors that influence mitochondrial oxidative phosphorylation rates are relatively unknown. In this report, we examine the effects of phosphoenolpyruvate (PEP), a glycolytic intermediate, on mitochondrial function. It is reported here that in rat heart mitochondria, PEP delays the onset of state 3 respiration in mitochondria supplied with either NADH-linked substrates or succinate. However, the maximal rate of state 3 respiration is only inhibited when oxidative phosphorylation is supported by NADH-linked substrates. The capacity of PEP to delay and/or inhibit state 3 respiration is dependent upon the presence or absence of ATP. Inhibition of state 3 is exacerbated in uncoupled mitochondria, with a 40% decrease in respiration seen with 0.1mM PEP. In contrast, ATP added exogenously or produced by oxidative phosphorylation completely prevents PEP-mediated inhibition. Mechanistically, the results support the conclusion that the main effects of PEP are to impede ADP uptake and inhibit NADH oxidation. By altering the NADH/NAD(+) status of mitochondria, it is demonstrated that PEP enhances succinate dehydrogenase activity and increase free radical production. The results of this study indicate PEP may be an important modulator of mitochondrial function under conditions of decreased ATP.     

Site of Action of the Cord Factor of Corynebacterium diphtheriae in Mitochondria

Trehalose-6,6'-dicorynomycolate (T66DCM), the cord factor of Corynebacterium diphtheriae, induced in vitro a swelling accompanied with a partially irreversible change of the mitochondrial membrane system in mouse liver. Preincubation of the mitochondrial suspension with T66DCM resulted in an inhibition of phosphorylation coupled to the oxidation of either succinate or a number of reduced nicotinamide adenine dinucleotide-linked substrates and a loss of respiratory control. T66DCM affected both electron transport and phosphorylation at coupling site II and uncoupled respiration and phosphorylation at coupling site III. T66DCM stimulated mitochondrial adenosine triphosphatase. The induction of adenosine triphosphatase by T66DCM and by 2,4-dinitrophenol was additive.     

Phosphorylation and uncoupled respiration in the tissue of rats during the development of homoiothermy

A marked increase in the rate of mitochondrial respiration, not coupled with ADP phosphorylation, was noted during the transformation of newborn poikilothermic animals into homoeothermic ones in the experiment on the rat tissue homogenates. Uncoupled respiration, as well as coupled one, is realized by the mitochondrial respiration chain, is observed upon oxidation of NADH, succinate, ascorbate and is expressed by a high rate of O2 consumption in the absence of added ADP. During ontogenesis, uncoupled respiration is activated to a greater extent in the heart and skeletal muscle and to a lesser extent in the liver and brown fat. The rates of phosphorylating oxidation of different substrates in tissue homogenates of animals from various age groups differ insignificantly. It is supposed that the postnatal development of homoeothermism in rats is ensured by the formation in many tissues of a system of uncoupled respiration, which takes part in heat production without preliminary ATP synthesis.     

Ziram, a sulfhydryl reagent and specific inhibitor of yeast mitochondrial dehydrogenases.

The fungicide zinc dimethyldithiocarbamate (ziram) is a sulfhydryl reagent which inhibits specifically the growth of the yeast Saccharomyces cerevisiae on nonfermentable substrates. In isolated mitochondria, the uncoupled as well as the state 3 oxidations of succinate, α-ketoglutarate, ethanol, and malate plus pyruvate are sensitive to ziram concentrations of 10 to 30 μm. The oxidations of isocitrate, of external NADH, of α-glycerophosphate, and of ascorbate plus tetramethylphenylenediamine exhibit a lower sensitivity to ziram. Succinate, α-ketoglutarate, and pyruvate dehydrogenases activities are 50% inhibited by concentration of ziram lower than 10 μm. At the same concentrations, neither the mitochondrial transports of succinate, ADP, or phosphate nor oxidative phosphorylation and adenosine triphosphatase activities are modified. The kinetic study of the inhibition by ziram of succinate dehydrogenase activity shows that ziram is noncompetitive with succinate and produces sigmoidal inhibitions of state 3 and of uncoupled oxidation of succinate by intact mitochondria. Inhibition of succinate:phenazine methosulfate oxidoreductase activity yields exponential kinetics. However sigmoidal-type inhibition is observed when succinate dehydrogenase activity is stimulated by ATP.     

Differential effects of N-acetylglutamate on citrulline synthesis by coupled and uncoupled mitochondria

When rats were placed on a low-protein (5%) diet for 24 h or less, liver mitochondrial acetylglutamate decreased rapidly, carbamyl phosphate synthetase (ammonia) and ornithine transcarbamylase decreased little, and carbamyl phosphate synthesis (measured as citrulline) by isolated mitochondria occurred at very low rates. The matrix acetylglutamate content of these mitochondria, whether coupled or uncoupled, was increased similarly by preincubating them with added acetylglutamate, but citrulline synthesis increased from less than 1 to 2.3 nmol min-1 mg-1 in the coupled state, and from less than 1 to 35 nmol min-1 mg-1 in the uncoupled state. However, when coupled mitochondria were incubated with the substrates required for the synthesis of acetylglutamate in the matrix, citrulline synthesis increased to 48 nmol min-1 mg-1; this rate was similar to that of mitochondria from control rats (fed a normal diet). When mitochondria from controls were incubated with up to 5mM acetylglutamate, citrulline synthesis by coupled mitochondria was increased by 10 to 40%, while synthesis by uncoupled mitochondria was 1.5 to 4 times higher than that observed with the coupled mitochondria; matrix acetylglutamate in both conditions rose to levels similar to those in the medium. The reason for the different behavior of carbamyl phosphate synthetase (ammonia) in coupled and uncoupled mitochondria was not apparent; neither oxidative phosphorylation nor ornithine transport were limiting in the coupled system. These observations are an example of the restrictions imposed upon enzymatic systems by the conditions existing in the mitochondrial matrix, and of the different behavior of carbamyl phosphate synthetase in situ and in solution. In addition, they show that conclusions about the characteristics of the enzyme in coupled mitochondria based on observations made in uncoupled mitochondria are not necessarily justified.     

Analysis of mutations that uncouple transport from phosphorylation in enzyme IIGlc of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.

Mutations that uncouple glucose transport from phosphorylation were isolated in plasmid-encoded Escherichia coli enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The uncoupled enzymes IIGlc were able to transport glucose in the absence of the general phosphoryl-carrying proteins of the PTS, enzyme I and HPr, although with relatively low affinity. Km values of the uncoupled enzymes IIGlc for glucose ranged from 0.5 to 2.5 mM, 2 orders of magnitude higher than the value of normal IIGlc. Most of the mutant proteins were still able to phosphorylate glucose and methyl alpha-glucoside (a non-metabolizable glucose analog specific for IIGlc), indicating that transport and phosphorylation are separable functions of the enzyme. Some of the uncoupled enzymes IIGlc transported glucose with a higher rate and lower apparent Km in a pts+ strain than in a delta ptsHI strain lacking the general proteins enzyme I and HPr. Since the properties of these uncoupled enzymes IIGlc in the presence of PTS-mediated phosphoryl transfer resembled those of wild-type IIGlc, these mutants appeared to be conditionally uncoupled. Sequencing of the mutated ptsG genes revealed that all amino acid substitutions occurred in a hydrophilic segment within the hydrophobic N-terminal part of IIGlc. These results suggest that this hydrophilic loop is involved in binding and translocation of the sugar substrate.     

Signal transduction through the T cell receptor-CD3 complex. Evidence for heterogeneity in receptor coupling

T cell receptor-CD3 complex (TCR-CD3)-mediated signal transduction was analyzed in HPB-ALL and Jurkat T cell lines. Both cell lines express high levels of TCR-CD3 complex on the cell surface, but provide different model systems for TCR-CD3 signaling in T cells. Jurkat responds with both inositol phosphate generation and intracellular Ca2+ mobilization after triggering of TCR-CD3, whereas TCR-CD3 triggering of HPB-ALL induces Ca2+ mobilization without detectable inositol phosphate generation. By employing a permeabilized cell system, we show that the HPB-ALL line expressed normal levels of Ca2(+)-induced phospholipase C activity. However, the TCR-CD3 on this cell line seems to be uncoupled from phospholipase C activation. In agreement with this result we also show, by analysis of protein kinase C-dependent phosphorylation of three distinct substrates, that TCR-CD3 in HPB-ALL is apparently uncoupled from protein kinase C activation. These findings may have implications for understanding signal-transducing pathways in T cells at various stages of differentiation.     

Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase

The hydroxylation of proline and lysine residues by the collagen hydroxylases is coupled with a stoichiometric decarboxylation of 2-oxoglutarate. Ascorbate is virtually a specific requirement for these enzymes, but previous studies have demonstrated that it is not consumed during most catalytic cycles. Prolyl 4-hydroxylase and lysyl hydroxylase are known also to catalyze an uncoupled decarboxylation of 2-oxoglutarate in the absence of the peptide substrate. It is shown here that, unlike the complete hydroxylation reaction, the uncoupled decarboxylation reaction involves stoichiometric ascorbate consumption. This stoichiometric ascorbate consumption was also seen when the rate of the uncoupled prolyl 4-hydroxylase reaction was enhanced by the addition of poly(L-proline). Since collagen hydroxylases may catalyze occasional uncoupled reaction cycles even in the presence of the peptide substrates, the main function of ascorbate in these reactions in vivo is suggested to be that of reactivating the enzymes after such uncoupled cycles.     

Substitution of an extracellular cysteine in the beta 2-adrenergic receptor enhances agonist-promoted phosphorylation and receptor desensitization

We constructed and expressed in a permanent cell line a beta 2-adrenergic receptor with a valine substitution for cysteine 184 of the second putative extracellular loop. The mutant receptor was partially uncoupled from adenylyl cyclase with impaired ability to form the high affinity agonist-receptor-G protein complex, yet displayed more rapid and extensive agonist-induced desensitization. The enhanced desensitization was accompanied by increased agonist promoted, but not cAMP promoted, receptor phosphorylation in intact cells. Thus, not only is impaired desensitization associated with decreased phosphorylation, as we have shown with several mutant beta 2-adrenergic receptors recently, but enhanced desensitization is accompanied by increased agonist promoted receptor phosphorylation. In the case of this cysteine mutant, this may be due to the greater accessibility of the uncoupled receptor for phosphorylation by the beta-adrenergic receptor kinase.     

CORTISONE-INDUCED ALTERATIONS IN MITOCHONDRIAL FUNCTION AND STRUCTURE

The effects of cortisone treatment on oxygen consumption, oxidative phosphorylation, and fine structure of rat liver mitochondria have been studied. Male rats weighing 125 g were treated for 6 days with 5 mg of cortisone acetate or isotonic saline. On the 7th day, sections of liver were excised and processed for light and electron microscopy. Mitochondrial respiration and oxidative phosphorylation were studied with mitochondria isolated from these livers. Cortisone treatment is responsible for a 14–40% decrease in the amount of oxygen consumed per mg of mitochondrial protein when succinate, α-ketoglutarate, or β-hydroxybutyrate are used as substrates, or with ascorbate and N,N,N¹,N¹-tetramethyl p-phenylenediamine as electron donors. In addition, oxidative phosphorylation is uncoupled with a lowering of the P:O ratios. Randomly selected liver cells have been analyzed by quantitative morphometric techniques. The average mitochondrial volume is increased fourfold in the peripheral and midzonal regions with a commensurate decrease in the number of mitochondria per cell. These alterations are present throughout the hepatic lobule, but are most marked in midzonal cells. The total mitochondrial volume per cell and the per cent of the total cytoplasmic volume occupied by mitochondria remains relatively unaltered, as does the total amount of cristae surface per cell. While the mitochondria are enlarged, they are not "swollen." The relationships between the steroid hormone treatment and the alterations in mitochondrial function and structure are discussed.     

Effect of rubomycin, carminomycin and adriamycin on respiration in liver mitochondria in various metabolic states, respiratory control and ADP/O ratio

A comparative study on the effects of antitumour antibiotics of the anthracycline group (rubomycin, carminomycin and adriamycin) on respiration and oxidative phosphorylation in liver mitochondria in various metabolic states has been carried out for the first time. It was shown that the antibiotics under study cause partial inhibition of mitochondrial state 3 respiration, which is eliminated by an uncoupler. Treatment of liver mitochondria with the antibiotics decreases the ADP/O and respiratory control values and stimulates state 4 respiration. The latter is partly inhibited by oligomycin. The uncoupled respiration is decelerated in the presence of the antibiotics. Under these conditions the oxidation of succinate is inhibited by lower concentrations of the antibiotics than that of NAD+-dependent substrates. It was shown that the maximal activity is exerted by the most polar agent carminomycin, while the hydrophobic rubomycin is the least active. The experimental results are discussed in terms of the toxic effect of antitumour antibiotics.     

Interaction of a synthetic polyanion with rat liver mitochondria

The effect of a polyanion (a copolymer of methacrylate, malaete and styrene in a 1:2:3 proportion with an average molecular weight of 10 000) on respiration, ATPase activity and ADP/ATP exchange activity of rat liver mitochondria and submitochondrial particles has been studied.The polyanion (at 17–150 μg/ml concentration, 100 μg polyanion corresponding to 0.83 μequiv. of carboxylic groups) inhibits the oxidation of succinate and NAD-linked substrates in state 3 in a concentration-dependent manner. The extent of this inhibition can be decreased by elevating the concentration of ADP. State 4 respiration is not affected by the polyanion. It has also a slight inhibitory effect on the oxidation of the above mentioned substrates in the uncoupled state (a maximum inhibition of 37% at 166 μg/ml polyanion concentration), which is unaffected by ADP. The strong inhibition of state 3 respiration can be relieved by 2,4-dinitrophenol to the low level observed in the uncoupled state. Ascorbate+TMPD oxidation is slightly inhibited in state 3, while it is not inhibited at all in the uncoupled state.The polyanion, depending on its concentration, strongly inhibits also the DNP-activated ATPase activity of mitochondria (50% inhibition at 40 μg/ml polyanion concentration).The ATPase activity of sonic submitochondrial particles is also inhibited. However, this inhibition is incomplete (reaching a maximum of 65%) and higher concentrations of the polyanion are required than to inhibit the ATPase activity of intact mitochondria.The polyanion inhibits the ADP/ATP translocator activity of mitochondria, measured by the “back exchange” of [2-³H]ADP. After a short preincubation of the mitochondria with the polyanion, the concentration dependence of the inhibition by the polyanion corresponds to that of the DNP-activated ATPase activity of intact mitochondria.It is concluded that, in intact mitochondria, the polyanion has at least a dual effect, i.e. it partially inhibits the respiratory chain between cytochrome b and cytochrome c, and strongly oxidative phosphorylation by blocking the ADP/ATP translocator.     

Amaranth seed oil: Effect of oral administration on energetic functions of rat liver mitochondria activated with adrenaline

Respiration parameters of liver mitochondria (MCh) in rats fed with amaranth seed oil for 3 weeks have been evaluated. Thirty minutes before decapitation, adrenaline was injected intraperitoneally at a low dose (350 μg/kg body weight) to both control and experimental animals. It was shown that in animals that were injected with adrenaline and did not receive oil, the rate of phosphorylating respiration increased by 32% and phosphorylation time decreased by 22% upon oxidation of succinate; upon oxidation of α-ketoglutarate in the presence of the succinate dehydrogenase inhibitor malonate, phosphorylating respiration was activated by 23%. The respiration of MCh upon oxidation of succinate + glutamate and α-ketoglutarate in the absence of malonate was not affected by adrenaline. The intake of oil markedly activated almost all parameters of mitochondrial respiration in experimental rats upon oxidation of all above-listed substrates in both coupled and uncoupled MCh. However, phosphorylation time was close to the control value (upon oxidation of succinate) or increased (upon oxidation of α-ketoglutarate in the presence and absence of malonate). The injection of adrenaline to animals receiving oil did not affect the oil-activated respiration of MCh oxidizing the substrates used; however, phosphorylation time in all groups of animals decreased. Ca²⁺ capacity of MCh in rats receiving amaranth oil did not change. Thus, our data show that feeding of rats with amaranth oil activates mitochondrial respiration and prevents MCh hyperactivation induced by adrenaline.     

Uncoupling of energy-linked functions of corn mitochondria by linoleic Acid and monomethyldecenylsuccinic Acid

Linoleic acid and monomethyldecenylsuccinic acid were tested as uncoupling agents for energy linked functions of corn mitochondria. 2,4-dinitrophenol was used as a standard for comparison. Both compounds uncoupled oxidative phosphorylation, released oligomycin-blocked respiration, and accelerated adenosine triphosphatase. Linoleic acid uncoupled calcium-activated phosphate accumulation and the increase in light scattering that accompanies the accumulation. Unlike dinitrophenol, linoleic acid at 0.1 mm had a destructive effect on membrane semipermeability. Kinetic studies indicated that dinitrophenol and linoleic acid compete with phosphate for active sites in oxidative phosphorylation.     

Polarographic observation of substrate-level phosphorylation and its stimulation by acetylcholine

Substrate-level phosphorylation was observed under the conditions optimal for this process and opposite to those for oxidative phosphorylation. Polarographic registration of Ca2+ stimulated alpha-ketoglutarate oxidation and self-inhibition of uncoupled alpha-ketoglutarate (KG) oxidation was used. Acetylcholine (ACh) administration stimulated KG oxidation and substrate-level phosphorylation in isolated mitochondria. These effects are stronger in tissues with a higher level of endogenous acetylcholine, such as guinea pig liver vs rat liver and pancreas vs liver. The specific stimulation of KG oxidation by ACh is related to a decrease of succinate oxidation and is contrary to the specific stimulating effect of adrenaline on succinate oxidation. Therefore the existence of reciprocal hormone-substrate-nucleotide systems is suggested. The described set of conditions optimal for substrate-level phosphorylation observation by polarographic registration of respiration is as convenient as the ADP test for the investigation of oxidative phosphorylation.     

Phosphorylation in Hydrogen Bacteria

The electron-transport system of cell-free extracts obtained from Hydrogenomonas H-20 has been studied with particular reference to phosphorylation associated with the oxyhydrogen reaction. Cell-free preparations of this organism exhibit oxidative phosphorylation with hydrogen and succinate as electron donors. This activity could be uncoupled with a number of agents. Ratios of phosphorylative activity to oxidative activity observed varied from 0.2 to 0.7. Factors affecting the efficiency of phosphorylation were examined. Inhibitor and spectrophotometric studies indicated that phosphorylation with hydrogen as electron donor occurs exclusively at a site in an abbreviated electron transport chain between H(2) and cytochrome b. The possible occurrence of a cytochrome b oxidase and the requirement for a quinone are discussed, as well as the correlation between the abbreviated pathway and the energy generation by the cell. Evidence is presented which indicates that nicotinamide adenine dinucleotide does not participate in the hydrogen oxidation path which is coupled to adenosine triphosphate formation.     

Isolation of mutants of Escherichia coli uncoupled in oxidative phosphorylation using hypersensitivity to streptomycin.

Mutants of Escherichia coli, harbouring the uncA401 or uncB402 alleles, were found to take up streptomycin more rapidly than the coupled parent strains. The increased rate of uptake results in greater sensitivity of the uncoupled strains, compared to the parent strains, to low concentrations of streptomycin. Studies with unc+ revertants showed that hypersensitivity to streptomycin is attributable to the mutation causing uncoupling. The uptake of streptomycin in an unc- strain is abolished by addition of the chemical uncoupler carbonylcyanide m-chlorophenylhydrazone. The phenotype of hypersensitivity to streptomycin can be used as a selection procedure for the isolation of uncoupled strains. In an experiment reported here, nine out of 12 strains isolated as being sensitive to streptomycin (at 2.5 micrograms/ml), were found to be unable to grow on succinate as a sole source of carbon. Five of the nine Suc- strains were found to be uncoupled in oxidative phosphorylation, and two of the five uncoupled strains lacked Mg2+-ATPase activity. The mutations causing uncoupling were cotransducible with the ilv genes.     

A (Ca2+, Mg2+)-ATPase activity in plasma membrane fragments isolated from squid nerves

A (Ca2+, Mg2+)-ATPase activity and a (Ca2+, Mg2+)-dependent phosphorylation from ATP have been found in plasma membrane fragments from squid optical nerves under conditions where contamination by intracellular organelles is unlikely. The properties of this (Ca2+, Mg2+)-ATPase activity are almost identical to those of the ATP-dependent uncoupled Ca2+ efflux observed in dialyzed squid giant axons. This gives further support to the notion that the mechanism responsible for maintaining the low levels of ionized Ca concentration in nerves at rest is not a Na+-Ca2+ exchange system but an ATP-driven uncoupled Ca2+ pump.     

Oxidative phosphorylation in mitochondria isolated from small intestinal epithelium of thiamphenicol-treated rats and control rats

Treatment of rats with thiamphenicol in a dose of 125 mg/kg per day for 60–64 h causes specific inhibition of mitochondrial protein synthesis, leading to a drastic decrease of the cytochrome c oxidase activity in intestinal epithelium. At the same time the mitochondrial ATPase activity becomes resistant to inhibition by oligomycin. Experiments with isolated intestinal mitochondria revealed that respiration in state 3 is diminished by 55% with succinate (5 mM) and by 40% with pyruvate (10 mM) plus L-malate (2 mM) as the substrates, both as compared to intestinal mitochondria isolated from control rats. P : O ratios as well as respiratory control indices are comparable in the two groups of animals. Uncoupled respiration is inhibited by 35% with succinate as the substrate, while the succinate cytochrome c reductase activity remains unaltered. No inhibition of uncoupled respiration with pyruvate plus L-malate as the substrates was observed. The ATP-P_i exchange activity in the mitochondria from the treated animals is diminished by about 75%. It is concluded that in the mitochondria of the treated animals the inhibition of the coupled respiration (state 3) is caused by the limitation of the ATP-generating capacity and that electron transport is rate limiting only with the rapidly oxidized substrates such as succinate, if respiration is uncoupled.