Effect of the Intravenous Anesthetic 2,6-Diisopropylphenol on Respiration and Energy Production by Rat Brain Synaptosomes

The sensitivity of the mitochondrial energy production system to propofol (DPP) has been investigated in rat brain synaptosomes. DPP at 0.8 mM concentration produced a partial inhibition of coupled respiration, an apparent decrease of the oxygen uptake stimulation induced by CCCP and a full inhibition of the mitochondrial ATP production by synaptosomes. Higher concentrations of DPP (1 mM) fully abolish uncoupler-dependent stimulation and at 1.3 mM DPP also coupled respiration is completely blocked. Similar results were obtained when dinitrophenol replaced CCCP and phenol or propylbenzene replaced DPP. The presence of the alkyl residues seems critical for the DPP effect. In the presence of 30 mM glutamate both respiration and ATP production are enhanced but DPP effects are similar to those obtained in the absence of glutamate.     

Energy metabolism of the rat renal cortex, as measured by incubation in various substrates

Different substrate mixtures were investigated for their effect on energy metabolism using sections of the rat renal cortex. Simultaneous determination of adenine nucleotide concentrations and determination of the damage quotient of oxidative phosphorylation proved to be appropriate parameters for selecting substrate mixtures that have a favorable effect on energy metabolism. A mixture of albumin (1 mM) and octanoate (5.6 mM) with electrolytes proved to be adequate. The extent of oxygen consumption (60%) and 14CO2 formation (75%) argues in favor of the metabolization of this mixture; a damage quotient of 23% and general constancy of the concentration of high-energy compounds render prospective their testing in animal experiments. Addition of dicarbosylic acids increase the antimycin A resistant oxygen consumption without any energy conservation being demonstrable. Therefore, these substrates should not be used for conservation.     

The effect of glucose and glutamine on the intracellular nucleotide pool and oxygen uptake rate of a murine hybridoma

The effects of media concentrations of glucose andglutamine on the intracellular nucleotide pools andoxygen uptake rates of a murine antibody-secretinghybridoma cell line were investigated. Cells takenfrom mid-exponential phase of growth were incubated inmedium containing varying concentrations of glucose(0–25 mM) and glutamine (0–9 mM). The intracellularconcentrations of ATP, GTP, UTP and CTP, and theadenylate energy charge increased concomitantly withthe medium glucose concentration. The total adenylatenucleotide concentration did not change over a glucose concentration range of 1–25 mM but therelative levels of AMP, ADP and ATP changed as theenergy charge increased from 0.36 to 0.96. Themaximum oxygen uptake rate (OUR) was obtained in thepresence of 0.1–1 mM glucose. However at glucoseconcentrations >1 mM the OUR decreased suggestinga lower level of aerobic metabolism as a result of theCrabtree effect.A low concentration of glutamine (0.5 mM) caused asignificant increase (45–128%) in the ATP, GTP,CTP, UTP, UDP-GNac, and NAD pools and a doubling ofthe OUR compared to glutamine-free cultures. Theminimal concentration of glutamine also caused anincrease in the total adenylate pool indicating thatthe amino acid may stimulate thede novosynthesis of nucleotides. However, all nucleotidepools and the OUR remained unchanged within the rangeof 0.5–9 mM glutamine.Glucose was shown to be the major substrate forenergy metabolism. It was estimated that in thepresence of high concentrations of glucose (10–25 mM),glutamine provided the energy for the maintenance ofup to 28% of the intracellular ATP pool, whereas theremainder was provided by glucose metabolism.(Author for correspondence; E-mail:     

Oxidative phosphorylation supported by an alternative respiratory pathway in mitochondria from Euglena

The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline-N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane potential in mitochondria isolated from dark-grown Euglena cells was determined. With L-lactate as substrate, ATP synthesis was partially inhibited by antimycin, but the other four inhibitors completely abolished the process. Cyanide also inhibited the antimycin-resistant ATP synthesis. Membrane potential was collapsed (<60 mV) by cyanide and stigmatellin. However, in the presence of antimycin, a H(+)60 mV) that sufficed to drive ATP synthesis remained. Cytochrome c reductase, with L-lactate as donor, was diminished by antimycin and myxothiazol. Cytochrome bc(1) complex activity was fully inhibited by antimycin, but it was resistant to myxothiazol. Stigmatellin inhibited both L-lactate-dependent cytochrome c reductase and cytochrome bc(1) complex activities. Respiration was partially inhibited by the five inhibitors. The cyanide-resistant respiration was strongly inhibited by diphenylamine, n-propyl-gallate, salicylhydroxamic acid and disulfiram. Based on these results, a model of the respiratory chain of Euglena mitochondria is proposed, in which a quinol-cytochrome c oxidoreductase resistant to antimycin, and a quinol oxidase resistant to antimycin and cyanide are included.     

Effects of Diltiazem on Bioenergetics, K+ Gradients, and Free Cytosolic Ca2+ Levels in Rat Brain Synaptosomes Submitted to Energy Metabolism Inhibition and Depolarization

Diltiazem was able to decrease the oxygen consumption rate and lactate production in synaptosomes isolated from rat forebrains, both under control and depolarized (40 microM veratridine) conditions, starting from a concentration of 250 microM. This effect was particularly evident when synaptosomes were depolarized by veratridine. This depolarization-counteracting action was evident also when transplasma membrane K+ diffusion potentials were measured after depolarization induced by veratridine and by rotenone with a glucose shortage. The concentrations of ATP, phosphocreatine, and creatine were less sensitive to diltiazem action. The concentration/response relationships were the same as those found for the oxygen consumption were the same as those found for the oxygen consumption rate, lactate production, and K+ diffusion potentials. The effects of 0.5 mM diltiazem in counteracting inhibition of energy metabolism induced by rotenone without glucose were no longer detectable when either Ca2+ or Na+ was absent from the incubation medium of synaptosomes. Diltiazem at the same concentrations (starting from 250 microM) was able to inhibit both the veratridine-induced and the rotenone-without-glucose-induced increase in intrasynaptosomal free Ca2+ levels evaluated with the fluorescent probe quin2. The results are discussed in view of a possible effect of diltiazem on voltage-dependent Na+ channels and the possibility of utilizing this approach for counteracting neuronal failure due to derangement of energy metabolism or hyperexcitation.     

Role of Oxidative Metabolism in the Energy Suppply of Active Potassium Transport in Erythrocytes of the Lamprey Lampetra fluviatilis

To study role of glycolysis and oxidative metabolism in providing active transport of monovalent cations, isolated erythrocytes of the lamprey Lampetra fluviatlis were incubated at 20°C in the presence of various metabolic inhibitors. The active (ouabain-sensitive) K⁺ (⁸⁶Rb) influx into erythrocytes did not change after cell incubation for 1–2 h in the absence of glucose or in the presence of 10 mM deoxy-D-glucose or 1 mM monoiodoacetate. Inhibitors of oxidative phosphorylation (antimycin A, rotenone, sodium azide, cyanide) produced a significant decrease (on average, by 74% ) in the active K⁺ transport in the lamprey erythrocytes. All blockers of oxidative phosphorylation produced the same degree of inhibition of the K⁺ transport after the cell pre-incubation with them for 30 and 60 min. In experiments with rotenone, the K⁺ influx was reduced statistically significantly as early as in 5 min of cell incubation and reached a maximal effect after 10–20 min. The intracellular ATP content in erythrocytes decreased by 17, 37, and 45% after 5, 10, and 20 min of cell incubation with rotenone, respectively. The active K⁺ transport in the lamprey erythrocytes is most likely to be closely associated with the intracellular ATP concentration. The data obtained indicate that the energy supply of the Na,K-pump in the lamprey erythrocytes is due exclusively to oxidative phosphorylation processes.     

Effects of the Inhibitory Protein of Ethylene Synthesis on ATP Levels in Mung Bean Hypocotyl Segments

The inhibitory protein of ethylene synthesis purified from mungbean seeds reduced ATP levels in mung bean hypocotyl segments.When the segments were incubated with 0.5mM IAA for 6 hr toinduce ethylene-producing activity, the presence of the inhibitoryprotein suppressed the ethylene production and ATP content inthe tissue about 82 and 60%, respectively. Similar suppressiveeffects were also observed for endogenous ethylene productionand ATP contents in tissue not treated with IAA. (Received June 20, 1981; Accepted October 24, 1981)     

Effect of dichloroacetate on acetyl-CoA content and acetylcholine synthesis in rat brain synaptosomes

In potassium-depolarized synaptosomes Ca²⁺ inhibited oxidation of pyruvate (30%) and decreased the level of acetyl-CoA in intrasynaptosomal mitochondria (32%). On the other hand, Ca²⁺ facilitated provision of acetyl-CoA to synaptoplasm, since under these condition no change of synaptoplasmic acetyl-CoA and twofold stimulation of acetylcholine synthesis were found. However, in Ca²⁺-activated synaptosomes both synaptoplasmic acetyl-CoA and acetylcholine synthesis were suppressed by 1 mM (–)hydroxycitrate by 27 and 29%, respectively. It was not the case in resting synaptosomes. Dichloroacetate (0.05 mM) partially reversed the inhibitory effect of Ca²⁺ on pyruvate metabolism in synaptosomes and whole brain mitochondria. In Ca²⁺-stimulated synaptosomes, the dichloroacetate overcame suppressive effects of (–)hydroxycitrate on the level of synaptoplasmic acetyl-CoA and acetylcholine synthesis, but not on citrate clevage. It is concluded that dichloroacetate may improve the metabolism of acetylcholine in activated cholinergic terminals by increasing the production of acetyl-CoA in mitochondria and increasing its provision through the mitochondrial membrane to synaptoplasm by the transport system, independent of the ATP-citrate lyase pathway.     

Relation between energy metabolism in mitochondria and conversion of 18 hydroxycorticosterone to aldosterone in adrenals

The purpose of this study was to see whether there was any link between conversion of 18 hydroxycorticosterone to aldosterone and mitochondrial energy metabolism. In vitro incubations of duck adrenal mitochondria with 18 OH B were used in this study. Results show that 18 oxidation is inhibited by compounds blocking electron transport (antimycin A, cyanide, rotenone, amytal). Inhibition induced by cyanide and antimycin A is reversed with ATP. 2,4 dinitrophenol, oligomycin and DCCD inhibit 18 oxidation but guanidine stimulate this reaction. Thus aldosterone synthesis from 18 OH B depends on energy metabolism in mitochondria. This is a very new aspect related to the last step of aldosterone synthesis.     

THE MECHANISM OF ACTION OF β-BUNGAROTOXIN

—β-Bungarotoxin, a presynaptically-acting polypeptide neurotoxin, caused an efflux from synaptosomes of previously accumulated γ-aminobutyric acid and 2-deoxy-d -glucose. The toxin-induced efflux of γ-aminobutyric acid occurred by a Na⁺ -dependent process while that of 2-deoxyglucose was Na⁺ -independent. These effects were also produced by treating synaptosomes with low molecular weight compounds, including fatty acids, that inhibit oxidative phosphorylation. After incubation with β-bungarotoxin, synaptosomes exhibited increased production of ¹⁴CO₂ from [U-¹⁴C]glucose and decreased ATP levels. β-Bungarotoxin treatment of various subcellular membrane fractions caused the production of a factor that uncoupled oxidative phosphorylation when added to mitochondria. Mitochondria from toxin-treated brain tissue exhibited a limitation in the maximal rate of substrate utilization. We conclude that β-bungarotoxin acts by inhibiting oxidative phosphorylation in the mitochondria of nerve terminals. This inhibition accounts for the observed β-bungarotoxin effects on synaptosomes and at neuromuscular junctions. We suggest that the effects on energy metabolism result from a phospholipase A activity found to be associated with the toxin.     

Changes induced by caffeine, theophylline and theobromine on calcium uptake, respiration and ATP levels in rat-liver mitochondria

1. The inhibition of calcium uptake and cellular respiration depend on the concentration of the compounds as shown by the concentration-effect curves. 2. The concentrations at which 50% inhibition of the transport of calcium takes place (caffeine 45 mM, theophylline 12 mM, theobromine, 4 mM) do not coincide exactly with those that produce the same effect on cellular respiration (caffeine 60 mM, theophylline 22 mM, theobromine 8 mM). 3. ATP concentrations under different conditions were also determined; a decrease in their value induced by the drugs was observed. No significant differences were observed, however, between the effect produced by the methyl-xanthines. 4. These findings suggest that these compounds are able to affect in some way the maintenance of energy gradients linked to the effects studied.     

Transmembrane gradient of K+ ions as an energy source in the yeast Saccharomyces carlsbergensis

In the presence of 100 mM glucose antimycin A inhibits the respiration of the yeast S. carlsbergensis by 94%, but does not affect the K+ efflux, Mn2+ influx or the synthesis of high molecular weight polyphosphate (HPP). Therefore phosphorylation at the respiratory chain level is not involved in HPP synthesis or Mn2+ accumulation. Zn2+ similar to Mn2+ induces K+ efflux and HPP synthesis, while Co2+ and Ni2+ fail to produce these effects. The extracellular K+ (1-5 mM KCl) completely inhibits the HPP synthesis and reduces Mn2+ uptake by 40%. NaCl (60 mM) inhibits the HPP synthesis by 28%. Nigericin, candicidin and FCCP plus valinomycin completely prevent the HPP synthesis. The prolonged accumulation of Zn2+ and Mn2+ is accompanied by HPP conversion into low molecular weight polyphosphate (LPP). The HPP synthesis in response to the K+ efflux may be regarded as a specific regulatory mechanism, which increases the energy efficiency of yeast metabolism.     

Is adenosine a second metabolic substrate for human red blood cells?

Adenosine is present in the micromolar range in human plasma. In this study, metabolism of adenosine, which was maintained between 0.62 +/- 0.03 and 2.92 +/- 0.43 microM by means of a continuous infusion using a Harvard infusion pump, was investigated in human red blood cells. It was found that lactate production increases linearly as the adenosine concentration was raised. Cells infused with an average adenosine concentration of 2 microM produced lactate comparable to that produced by 5 mM glucose. The extent to which ATP concentration is maintained by adenosine also depends on its concentration. After a 4 h infusion with an average adenosine concentration of 0.7 microM, ATP content amounts to 75% of the glucose control. Raising the adenosine infusion concentration to 1.5 microM results in a full maintenance of ATP levels and at concentrations higher than 1.5 microM, adenosine produces a net synthesis of ATP. A net synthesis of ATP also occurs with adenosine concentration below 1.5 microM, if supplemented with glucose. In contrast, inosine infusion provides only a partial support of ATP and fails to produce a net synthesis of ATP in the presence of glucose. In addition, the presence of purine nucleoside and glucose together influence the metabolism of each other, depending on inorganic phosphate content (Pi). At a Pi concentration of 1 mM, the glucose consumption rate is reduced by approx. 25% by purine nucleoside infusion and vice versa. In sharp contrast, glucose consumption at 16 mM Pi is potentiated by adenosine. These findings suggest that plasma adenosine contributes significantly to human red cell energetics, even though it is present at a concentration several orders of magnitude lower than glucose.     

The respiration-linked limiting step of tumor cell transition from the non-cycling to the cycling state: Its inhibition by oxidizable substrates and its relationships to purine metabolism

The recruitment into the cycling state of resting Yoshida AH 130 hepatoma cells was studied with respect to its dependence on respiration in an experimental system wherein the overall energy requirement for this recruitment can be supplied by the glycolytic ATP. The G₁-S transition of these cells, unaffected by 2,4-dinitrophenol (DNP) at concentrations which uncouple the respiratory phosphorylation, is impaired either by blocking the electron flow to oxygen by antimycin A or by adding an excess of some oxidizable substrates, chiefly pyruvate and oxalacetate. An experimental analysis, focused on pyruvate activity, showed that the inhibition of cell recruitment into S is not related to the depressing effects of this substrate on aerobic glycolysis of tumor cells, nor is it modified by forcing, in the presence of DNP, pyruvate oxidation through the tricarboxylic acid cycle as well as the overall oxygen consumption. Addition of suitable concentrations of preformed purine bases (mainly adenine), completely removes the block of the G₁-S transition produced either by the excess of oxidizable substrates or by antimycin A. These findings indicate the existence of a respiration-linked step in purine metabolism, which restricts the above transition and is equally impaired by blocking the respiratory chain or by saturating it with an excess of reducing equivalents derived from unrelated oxidations. The inhibitory effects of pyruvate and antimycin A can be largely removed by the addition of folate and tetrahydrofolate, suggesting that the respiration-linked restriction point of tumor cell cycling involves the folate metabolism and its connections to purine synthesis.     

Control analysis of mitochondrial metabolism in intact hepatocytes: effect of interleukin-1beta and interleukin-6

Interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) are produced by hepatic nonparenchymal cells after systemic injury and have been reported to inhibit ATP synthesis in hepatocytes, which may contribute to hepatic dysfunction in inflammatory states. To elucidate the mechanisms of action of IL-1beta and IL-6 on hepatocellular ATP synthesis, we measured the oxygen uptake rate (OUR) and mitochondrial membrane potential (MMP) of stable hepatocyte cultures, and analyzed the dynamic MMP response following the addition of mitochondrial inhibitors (antimycin A and oligomycin) with a model of mitochondrial metabolism. IL-1beta reduced mitochondrial OUR coupled to ATP synthesis via inhibition of phosphorylation reactions which dissipate the MMP, including ATP synthesis and consumption. Furthermore, the ATP synthesis rate in cytokine-free and IL-1beta-treated hepatocytes was controlled primarily by phosphorylation reactions, which corresponds to a state where the ATP synthesis rate closely follows the cellular energy demand. Thus, IL-1beta-mediated effects on electron transport and substrate oxidation reactions are not likely to significantly impact on ATP synthesis. IL-6 did not reduce mitochondrial OUR coupled to ATP synthesis, but shifted the control for ATP synthesis towards processes which generate the MMP, indicating that IL-6 induces a metabolic state where cellular functions are limited by the mitochondrial energy supply.     

Iodoacetic acid-induced rigor in ileal longitudinal smooth muscle of guinea-pig

1. Ileal tensions to iodoacetic acid (IAA) develop when tissue ATP concentration falls below approximately 60-55% of the control. 2. As the IAA concentration is increased (0.1-10 mM), the ATP concentrations decrease rapidly, and both the time of the onset and the duration of the contraction shorten. 3. In the presence of lactate, IAA failed to decrease the tissue ATP concentration and did not develop tension. 4. The contraction to IAA developed in the presence of Ca2+ antagonist, D-600 or in Ca2(+)-free solution, however, onset time was prolonged. 5. These results suggest that the contraction to IAA is referred to as 'rigor' because it increases with decreasing tissue ATP concentration in ileum.     

Effects of probes of membrane potential on metabolism in synaptosomes

Effects of three probes for measuring membrane potential, tetraphenylphosphonium (TPP+), rhodamine 6G and 3,3'-dipropylthiocarbocyanine (diS-C3-(5)) on energy metabolism in synaptosomes were investigated. None of the three probes had any effect on lactate production in synaptosomes. TPP+ and rhodamine 6G did not inhibit the respiration of synaptosomes with pyruvate and succinate as exogenous substrate and were only weakly inhibitory with endogenous substrates. In contrast, diS-C3-(5) markedly inhibited the respiration of synaptosomes with glucose, pyruvate and endogenous substrates. All three probes reduced ATP content in synaptosomes and depolarized the membrane potential in synaptosomes with increasing concentrations of the probes. It is, therefore, preferable to estimate membrane potential with TPP+ or rhodamine 6G at their low concentrations where their effect on metabolism is negligible.     

Stimulation of dopamine synthesis and activation of tyrosine hydroxylase by phorbol diesters in rat striatum

In rat striatal synaptosomes, 4 beta-phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-dibutyrate (PDBu), two activators of Ca2+-phospholipid-dependent protein kinase (protein kinase C) increased dopamine (DA) synthesis measured by following the release of 14CO2 from L-[1-14C] tyrosine. Maximal stimulation (21-28% increase of basal rate) was produced by 0.5 microM PMA and 1 microM PDBu. 4 beta-Phorbol and 4 beta-phorbol 13-acetate, which are not activators of protein kinase C, were ineffective at 1 microM. PMA did not change the release of 14CO2 from L-[1-14C]DOPA. Addition of 1 mM EGTA to a Ca2+-free incubation medium failed to affect PMA stimulation. KC1 (60 mM) enhanced DA synthesis by 25%. Exposure of synaptosomes to either PMA or PDBu prior to KC1 addition resulted in a more than additive increase (80-100%) of DA synthesis. A similar synergistic effect was observed when the phorbol diesters were combined with either veratridine or d-amphetamine but not with forskolin and dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with phorbol diesters produced an activation on of tyrosine hydroxylase (TH) associated with a 60% increase of the Vmax and a decrease of the Km for the pterine cofactor 6-methyl-5,6,7,8-tetrahydropterin. These results indicate that protein kinase C participates in the regulation of striatal TH in situ and that its activation may act synergistically with DA releasing agents in stimulating DA synthesis.     

Cell de-energization prevents plasmid transformation of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: evidence for the requirement of ATP

The dependence of the yeast Saccharomyces cerevisiae transformation on energy requirement was studied. The inhibitory effect of sodium arsenate, used for the depletion of the intracellular ATP pool, was determined. Incubation of the yeast cells in 5 mM sodium arsenate diminished ATP accumulation by 50% and the transformation efficiency decreased by 65%. To discriminate between ATP produced by substrate level phosphorylation and oxidative phosphorylation, the inhibitory analysis of a mutant with defective mitochondria was performed. Sodium fluoride (10–50 mM), as inhibitor of glycolysis, elicited a concentration-dependent decrease in intracellular ATP levels in both parental and mutant cells. The equal transformation efficiency of the mitochondrial mutant and parental strain, in addition to experiments with oligomycin, demonstrated the independence of plasmid transformation on mitochondrial ATP synthesis. This is consistent with our hypothesis that yeast transformation efficiency is associated with ATP produced by substrate level phosphorylation.     

Glucose,Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals

Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate. Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes are able to use besides glucose and pyruvate also the substrates lactate, glutamate and glutamine to support their basal respiration. Veratridine was found to increase respiration supported by glucose, pyruvate, lactate and glutamine and FCCP was found to increase respiration supported by glucose, pyruvate and lactate. This was not the case when glutamate was the only energy substrate.