Relation between energy production and adenine nucleotide metabolism in human blood platelets

The relation between ATP production and adenine nucleotide metabolism was investigated in human platelets which were starved by incubation in glucose-free, CN^?-containing medium and subsequently incubated with different amounts of glucose. In the absence of mitochondrial energy production (blocked by CN^?) and glycogen catabolism (glycogen almost completely consumed during starvation), lactate production increased proportionally with increasing amounts of glucose. The generated ATP was almost completely consumed in the various ATP-consuming processes in the cell except for a fixed portion (about 7%) that was reserved for restoration of the adenylate energy charge. During the first 10 min after glucose addition, the adenine nucleotide pool remained constant. Thereafter, when the glycolytic flux, measured as lactate formation, was more than 3.5 μmol · min^?1 · 10^?11 cells, the pool increased slightly by resynthesis from hypoxanthine-inosine and then stabilized; at a lower flux the pool decreased and metabolic ATP and energy charge declined to values found during starvation. Between moments of rising and falling adenylate energy charges, periods of about 10 min remained in which the charge was constant and ATP supply and demand had reached equilibrium. This enabled comparison between the adenylate energy charge and ATP regeneration velocity. A linear relation was obtained for charge values between 0.4 and 0.85 and ATP regeneration rates between 0.6 and 3.5 ATP equiv. · min^?1 · 10^?11 cells. These data indicate that in starved platelets ATP regeneration velocity and energy charge are independent and that each appears to be subject to the availability of extracellular substrate.     

A critical appraisal of the association between energy charge and cell damage

The association between the energy charge and cellular damage caused by metabolic inhibitors was investigated in a cellular system of quiescent fibroblasts. The cell damage was assessed by the release of lactate dehydrogenase (LDH) which indicates a severe change of membrane integrity. Inhibition of glycolysis resulted in release of LDH when the energy charge decreased below 0.5 at an ATP level of 10% of the original level. If oxidative phosphorylation was inhibited, the energy charge decreased to 0.1-0.35 (dependent on the type of inhibitor) a long time before release of LDH, and no change occurred in the energy charge when release of LDH started. The ATP level was 0.5-2% of the original at this time. Even a decrease of the energy charge to 0.1 could be reversed to a normal level, and at the same time the morphological cellular changes were fully reversed and no release of LDH occurred. The conclusion is that no simple correlation between energy charge and cell survival exists. The different levels of ATP at which release of LDH started after inhibition of glycolysis and oxidative phosphorylation indicate a special role of glycolysis in maintaining the membrane function and integrity. This was emphasized by measuring the potassium loss of the cells which was much more marked after inhibition of glycolysis.     

Adenylate energy charge of rat and human cultured hepatocytes

Summary A simple and rapid method for the assay of adenine nucleotides (ATP, ADP, and AMP) was established to evaluate the adenylate energy charge (ATP+ADP/2)/(ATP+ADP+AMP) of cultured hepatocytes. The effects of inhibitors of glycolysis, fatty acid oxidation, or oxidative phosphorylation on the energy charge were examined. The energy charges of cultured hepatocytes in rats and human were almost identical and were maintained at a high level between 6 and 24 h after changing the media (rat: 0.908±0.008n=9, human: 0.918±0.014n=6, mean ± SD). Inhibition of glycolysis with sodium fluoride or oxidative phosphorylation with antimycin A irreversibly reduced both the adenine nucleotide contents and the energy charge. However, the inhibition of fatty acid oxidation with 2-tetradecylglycidic acid did not affect the nucleotide contents, and the energy charge only decreased transiently to recover within 8 h. When the inhibitor of oxidative phosphorylation was removed, the recovery in the energy charge preceded the recovery in the adenine nucleotide contents. These findings suggest that the adenylate energy charge is a more sensitive measure of the changes in energy metabolism than the adenine nucleotide contents. Furthermore, energy charge regulates adenine nucleotide contents in cultured hepatocytes. It is important to confirm that the high energy charge of the cultured hepatocytes is maintained when these cells are used for metabolic studies.     

Das Adenosinphosphat-System während Wachstum und Entwicklung von Acanthamoeba castellanii

The concentration of adenosine tri-, adenosine di-, and adenosine monophosphate in cells of Acanthamoeba castellanii was measured during the logarithmic growth phase and the stationary growth phase in which trophozoites were transformed into cysts. This developmental process was induced in three ways: by growth in nutrient medium to high cell density, by transferring cells in the logarithmic phase into a nutrient-free medium, and by mixing logarithmically growing cells with ethidium bromide. In all cases, encystment is accompanied with a reduction of total adenosine phosphate content to about 85%, mainly because of a depletion of cellular ATP. The value of the adenosine phosphate energy charge in logarithmically growing amoebae is 0.83. During development the energy charge becomes stabilized at different values (between 0.58 and 0.81), characteristic to the mode of encystation. A possible functional relationship between changes of the adenosine phosphate concentration and developmental processes of the amoeba is discussed.     

Energetics and Surface Properties of Pseudomonas putida DOT-T1E in a Two-Phase Fermentation System with 1-Decanol as Second Phase

The solvent-tolerant strain Pseudomonas putida DOT-T1E was grown in batch fermentations in a 5-liter bioreactor in the presence and absence of 10% (vol/vol) of the organic solvent 1-decanol. The growth behavior and cellular energetics, such as the cellular ATP content and the energy charge, as well as the cell surface hydrophobicity and charge, were measured in cells growing in the presence and absence of 1-decanol. Although the cells growing in the presence of 1-decanol showed an about 10% reduced growth rate and a 48% reduced growth yield, no significant differences were measured either in the ATP and potassium contents or in the energy charge, indicating that the cells adapted completely at the levels of membrane permeability and energetics. Although the bacteria needed additional energy for adaptation to the presence of the solvent, they were able to maintain or activate electron transport phosphorylation, allowing homeostasis of the ATP level and energy charge in the presence of the solvent, at the price of a reduced growth yield. On the other hand, significantly enhanced cell hydrophobicities and more negative cell surface charges were observed in cells grown in the presence of 1-decanol. Both reactions occurred within about 10 min after the addition of the solvent and were significantly different after killing of the cells with toxic concentrations of HgCl₂. This adaptation of the surface properties of the bacterium to the presence of solvents seems to be very similar to previously observed reactions on the level of lipopolysaccharides, with which bacteria adapt to environmental stresses, such as heat shock, antibiotics, or low oxygen content. The results give clear physiological indications that the process with P. putida DOT-T1E as the biocatalyst and 1-decanol as the solvent is a stable system for two-phase biotransformations that will allow the production of fine chemicals in economically sound amounts.     

Larger adenylate energy charge and ATP/ADP ratios in aerenchymatous roots of Zea mays in anaerobic media as a consequence of improved internal oxygen transport

Internal transport of O₂ from the aerial tissues along the adventitious roots of intact maize plants was estimated by measuring the concentrations of adenine nucleotides in various zones along the root under an oxygen-free atmosphere. Young maize plants were grown in nutrient solution under conditions that either stimulated or prevented the formation of a lysigenous aerenchyma, and the roots (up to 210 mm long) were then exposed to an anaerobic (oxygen-free) nutrient solution. Aerenchymatous roots showed higher values than non-aerenchymatous ones for ATP content, adenylate energy charge and ATP/ADP ratios. We conclude that the lysigenous cortical gas spaces help maintain a high respiration rate in the tissues along the root, and in the apical zone, by improving internal transport of oxygen over distances of at least 210 mm. This contrasted sharply with the low energy status (poor O₂ transport) in non-aerenchymatous roots.Abbreviation AEC adenylate energy charge     

Cell cycle regulation during development and dormancy in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus can enter into a state of metabolic dormancy, termed diapause, as a normal part of their development. In addition, these embryos can also survive for prolonged sojourns in the complete absence of oxygen. Dormant embryos support their metabolism using anaerobic metabolic pathways, regardless of oxygen availability. Dormancy in diapause is associated with high ATP and a positive cellular energy status, while anoxia causes a severe reduction in ATP content and large reductions in adenylate energy charge and ATP/ADP ratios. Most cells are arrested in the G₁/G₀ phase of the cell cycle during diapause and in response to oxygen deprivation. In this paper, we review what is known about the physiological and biochemical mechanisms that support metabolic dormancy in this species. We also highlight the great potential that this model holds for identifying novel therapies for human diseases such as heart attack, stroke and cancer.     

Glutamine synthetase regulation by energy charge in sunflower roots

Energy charge [(ATP) + ½ (ADP)]/[(ATP) + (ADP) + (AMP)] and glutamine synthetase activity (transferase reaction) of roots increase in a near congruent manner when decotyledonized sunflower plants (Helianthus annuus L. var. Mammoth Russian) are grown in nitrate for 9 days. Replacement of nitrate with ammonium for the final 2 days leads to a higher energy charge and increased enzyme activity. Similar correlations occur when nitrate plants are placed on a zero nitrogen regimen and when they are subjected to continuous darkness. A rank order correlation of 0.72 is obtained for all data. Control concepts such as adenylylation-deadenylylation and ammonium inhibition of enzyme synthesis are not supported by the data. Energy charge-enzyme activity plots support the view that glutamine synthetase of sunflower roots is subject to control by end products of glutamine metabolism. Alanine appears to exert a modulating effect on the regulation of glutamine synthetase by energy charge.     

MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation

In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2.     

The adenylate energy charge in marine phytoplantkon: The effect of temperature on the physiological state of Skeletonema costatum (Grev.) Cleve

The adenylate energy charge $\text{([ATP] +}\text{1}\text{2}\text{[ADP])}\text{[0ATP+ADP+AMP]}$ was measured in axenic batch cultures of Skeletonema costatum (Grev.) Cleve at 2°, 10°, 15°, 20°, 24° and 30°C. The results suggest that this eurythermal diatom is physiologically capable of adapting to the 28 °C range of temperature with little apparent difference in the potential energy available to the cell. In N-limited continuous cultures at 15 °C, the energy charge values were lower than those observed in batch culture by 0.2, implying nutrient stress may result in decreased intracellular chemical energy. The utilization of the adenylate energy charge as an indicator of physiological state is suggested.     

Rapid estimation of the energy charge from cell lysates using matrix-assisted laser desorption/ionization mass spectrometry: Role of in-source fragmentation

Nucleotides are key players in the central energy metabolism of cells. Here we show how to estimate the energy charge from cell lysates by direct negative ion matrix-assisted laser desorption/ionization mass spectrometry (MALDI–MS) using 9-aminoacridine as matrix. We found a high level of in-source decay of all the phosphorylated nucleotides, with some of them producing considerable amounts of adenosine-5′-diphosphate (ADP) fragment ions. We investigated the behavior of adenosine-5′-monophosphate (AMP), ADP, and adenosine-5′-triphosphate (ATP) as well as the cofactors coenzyme A (CoA) and acetyl-coenzyme A (ACoA) and nicotinamide adenine dinucleotides (NAD⁺ and NADH) in detail. In-source decay of these compounds depends strongly on the applied laser power and on the extraction pulse delay. At standard instrument settings, the 9-aminoacridine (9-AA) matrix resulted in a much higher in-source decay compared with 2,4,6-trihydroxyacetophenone (2,4,6-THAP). By adding ¹³C-labeled ATP to a cell lysate, we were able to determine the degree of in-source decay during an experiment. Analyzing a cell extract of the monocytic cell line THP-1 with [¹³C]ATP as internal standard, we were able to obtain values for the energy charge that were similar to those determined by a reference liquid chromatography electrospray ionization coupled to mass spectrometry (LC–ESI–MS) method.     

Control of hydrogen-dependent nitrogenase activity by adenylates and electron flow in heterocysts of Anabaena variabilis (ATCC 29413)

Hydrogen-dependent nitrogenase activity was studied in heterocysts, isolated from the filamentous cyanobacterium Anabaena variabilis (ATCC 29413). Hydrogen provides reductant and ATP for nitrogenase via linear electron flow through Photosystem I. This allows for regulation of nitrogenase activity by controlling the turnover of the photosystem. When nitrogenase activity was varied by changing either the light intensity or the supply of reductant (i.e., hydrogen) or by inhibition of photosynthetic electron transport by DBMIB, no rate-dependent changes in cellular ATP concentrations were observed. This homeostasis of ATP was perturbed by addition of metronidazole, acting as alternative electron sink to nitrogenase, and by uncoupling agents like FCCP, gramicidin and nigericin. Valinomycin (in presence of KCl) exerted little effect on nitrogenase activity and adenylate pool composition. Metronidazole increased and uncoupling agents decreased cellular ATP concentration, ATP/ADP ratio and energy charge. Inhibition of nitrogenase activity by metronidazole was caused by reductant limitation; inhibition by uncoupling agents was due to energy limitation. Control exerted on nitrogenase activity by ATP (energy limitation) was more pronounced at high rates of electron flow to nitrogenase than during reductant limitation. When cellular ATP synthesis was suboptimal due to partial uncoupling, the connection of phosphorylation and nitrogenase activity by electron transport allowed for homeostasis of ATP also at a lowered cellular concentration.     

Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions

The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.     

Changes in adenosine phosphates and energy charge in chloroplastic and nonchloroplastic compartments of wheat leaves (author's transl)]

Changes in the adenine nucleotides and energy charge (= (ATP)+1/2(ADP)/(AMP)+(ADP)+(ATP)) levels were studied in chloroplastic and non-chloroplastic compartments using non-aqueously isolated wheat leaves chloroplasts. The two adenine nucleotides pools (of chloroplasts and non-chloroplastic part of the cell), though distinct, are linked. This linkage substantiates an energy-rich bond exchange between the two compartments. When both photphosphorylation and oxidative phosphorylation occur simultaneously, energy charge takes high values, generally higher than 0.80. When neither oxidative phosphorylation nor photophosphorylation occur, energy charge is very low and takes values generally lower than 0.45. When one compartment alone produces approximately P, energy charge in the two compartments takes intermediate values which remain relatively high. Dark-light transition in nitrogen resulted in changes of the AMP, ADP and ATP levels which quickly reach a steady state. Chloroplast energy charge shifts rapidly from 0.45 to 0.75 in 10 s; after 1 min it reaches 0.86, a value that corresponds to a steady level. In the cytoplasm, energy charge changes from 0.44 to 0.71 in 1 min. Energy charge increase in the non-chloroplastic compartment substantiates an energy transfer from chloroplasts to the cytoplasm. On nitrogen-air transition in the dark, the cytoplasm energy charge reaches a steady level in 30 s. In chloroplasts, it also increases but slowly. There is indeed a transfer of energy from cytoplasm to chloroplasts. Darkening of the leaves in air causes a drastic and lasting drop of energy charge in the chloroplasts where it has a low value after 5 min in the dark. Then it increases again but slowly and is still lower than 0.70 after 10 min in the dark. Meanwhile, energy charge in cytoplasm keeps values higher than 0.75. Metabolic regulation by energy charge and control of adenine nucleotides level by adenylate kinase (EC 2.7.4.3) are discussed.     

AMP-activated protein kinase: the energy charge hypothesis revisited.

The AMP-activated protein kinase cascade is a sensor of cellular energy charge, and its existence provides strong support for the energy charge hypothesis first proposed by Daniel Atkinson in the 1960s. The system is activated in an ultrasensitive manner by cellular stresses that deplete ATP (and consequently elevate AMP), either by inhibiting ATP production (e.g., hypoxia), or by accelerating ATP consumption (e.g., exercise in muscle). Once activated, it switches on catabolic pathways, both acutely by phosphorylation of metabolic enzymes and chronically by effects on gene expression, and switches off many ATP-consuming processes. Recent work suggests that activation of AMPK is responsible for many of the effects of physical exercise, both the rapid metabolic effects and the adaptations that occur during training. Dominant mutations in regulatory subunit isoforms (gamma2 and gamma3) of AMPK, which appear to increase the basal activity in the absence of AMP, lead to hypertrophy of cardiac and skeletal muscle respectively.     

Close correlation between platelet responses and adenylate energy charge during transient substrate depletion

The relation between availability of metabolic energy and thrombin-induced platelet aggregation and secretion was investigated in a system of transient substrate depletion followed by restoration of ATP resynthesis. Substrate depletion induced a fall in the concentration of metabolic ATP and in the adenylate energy charge and a concurrent decline in aggregation and secretion of dense and α-granule contents. Restoration of energy generation completely restored the adenylate energy charge and restored aggregation and secretion, but led to incomplete recovery of the ATP concentration. A close correlation between the adenylate energy charge and aggregation and between the adenylate energy charge and the secretion of dense and α-granule contents could be demonstrated. No such correlation existed between these responses and the concentration of ATP. These results show that the adenylate energy charge monitors an energetic condition which is crucial for preservation of platelet aggregation and secretion of dense and α-granule contents.     

Relationship between ATP and energy charge during lethal metabolic stress of the marine isopod Cirolana borealis

The relationship between ATP and energy charge was studied in individuals of Cirolana borealis under heavy metabolic stress caused by anoxia or exposure to toluene. Prolonged anoxia led to a lowering of the ATP content to about 10% after 4 days, with a simultaneous decrease in energy charge to about 0.25. A lowering of the total adenylate pool reduced the fall in energy charge somewhat, but this effect was marked only in late anoxia when the individuals had become inactive. Exposure to 0.14 mM toluene for 8 days led to a similar decrease in ATP and energy charge. Exposure to 1.4 mM toluene for 24 h led to only slight changes in the adenine nucleotide pool, although the individuals became narcotized within a few hours. The energy charge associated with moribund individuals thus varied much. The mechanism of energy charge stabilization through reduction of the adenine nucleotide pool as ATP declined seemed to be of little significance for the survival of the individuals.     

Response of nucleoside diphosphate kinase to the adenylate energy charge

The reaction catalyzed by nucleoside diphosphate kinase responds to the energy charge of the adenylate pool. The velocity is maximal at a charge of 1.0, and decreases sharply with a decrease in the charge. This response may control the flow of phosphate from ATP into the other nucleotide pools and thus participate in the regulation of macromolecular synthesis by the energy level of the cell, as reflected in the charge of the adenylate pool.     

ATP content and adenylate energy charge ofBacillus stearothermophilus during growth

The ATP content and the adenylate energy charge of the thermophileBacillus stearothermophilus were determined during growth.Bacillus subtilis was used for comparison to determine whether there were differences in the ATP content and adenylate energy charge between a mesophile and a thermophile. Both the ATP content and the adenylate energy charge were lower in the thermophile than in the mesophile. These lower values may reflect a decreased activation energy required for the metabolic coupling when growth is occurring at the higher temperatures characteristic of the thermophile.     

Some thoughts on the evolutionary basis for the prominent role of ATP and ADP in cellular energy metabolism

The predominance of the adenosine triphosphate/adenosine diphosphate (ATP/ADP) couple in cellular phosphorylation reactions, including those that form the basis for cellular energy metabolism, cannot be explained on thermodynamic grounds since a variety of "high energy phosphate" compounds (including ADP itself) found in the cell would, based on thermodynamic considerations, be at least as effective as ATP in serving as a phosphoryl donor. How then did present-day organisms come to rely on the ATP/ADP couple as the principal mediator of phosphorylation reactions? The early appearance of adenine compounds in the prebiotic environment is suggested by experiments indicating that, relative to other purine or pyridimine compounds, adenine derivatives are preferentially synthesized under simulated prebiotic conditions (Ponnamperuma et al., 1963). In addition to the roles of adenine nucleotides in phosphorylation reactions, other adenine derivatives (e.g. Coenzyme A, flavin adenine dinucleotide, puridine nucleotides) are employed in a variety of metabolic roles. The principal function of the adenine moiety in these latter cases is in the binding of these derivatives to the relevant enzyme. The capability for binding of the adenine moiety appears to have arisen early in evolution and been exploited in a multitude of contexts, a suggestion consistent with observed similarities between the binding sites of several enzymes employing adenine derivatives as substrate. The early availability of suitable adenine compounds in the biosphere and development of complementary binding sites on cellular proteins, coupled with the expected advantages in having a limited number of metabolites as central mediators of endergonic and exergonic metabolism could readily have led to the observed pre-eminence of adenine nucleotides in cellular energy metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)