Steady-state Concentration Of ATP Research Articles

Polyphosphate affects cytoplasmic and chromosomal dynamics in nitrogen-starved Pseudomonas aeruginosa

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP's many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we quantify the motion of chromosomal loci and cytoplasmic tracer particles. In the absence of polyP and upon starvation, we observe a 2- to 10-fold increase in mean cytoplasmic diffusivity. Tracer particles reveal that polyP also modulates the partitioning between a "more mobile" and a "less mobile" population: Small particles in cells unable to make polyP are more likely to be "mobile" and explore more of the cytoplasm, particularly during starvation. Concomitant with this larger freedom of motion in polyP-deficient cells, we observe decompaction of the nucleoid and an increase in the steady-state concentration of ATP. The dramatic polyP-dependent effects we observe on cytoplasmic transport properties occur under nitrogen starvation, but not carbon starvation, suggesting that polyP may have distinct functions under different types of starvation.

Cytochrome c oxidase is activated by the oncoprotein Ras and is required for A549 lung adenocarcinoma growth

BackgroundConstitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX).ResultsWe found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice.ConclusionTaken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents.

Abstract 2825: Characterization of a novel small molecule antagonist of 6-phosphofructo-2-kinase that suppresses glucose metabolism and tumor growth

Abstract Over-expression of HIF-1α, activation of Ras and loss of p53 tumor suppressor function are associated with the development of human cancers and each of these genetic alterations converge on glycolysis by activating the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB). The PFKFB enzymes interconvert fructose-6-phosphate (F6P) and fructose-2,6-bisphosphate (F2,6BP) and F2,6BP is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme and control point in the glycolytic pathway. There are four PFKFB family members which are encoded by separate genes (PFKFB1-4) and characterized by distinct kinase:bisphosphatase ratios. The PFKFB3 family member is of particular interest since it has been found to be activated in human cancer cell lines and tumors, to be increased by hypoxic exposure via HIF-1α and by oncogenic Ras, and to be required for tumorigenic growth. We recently discovered a competitive antagonist of PFKFB3 (3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one [3PO]) that reduces the glucose uptake, F2,6BP, lactate secretion, steady-state concentration of ATP, and growth of multiple transformed cell lines. Using a rationale design based approach, we have synthesized derivatives of 3PO and identified a compound (PFK-015) that: (i) more potently inhibits recombinant PFKFB3 (IC50:110 nM); (ii) inhibits PFKFB3 activity in cancer cells (IC50: 20 nM); (iii) is not a Pgp substrate as determined by transport and cell permeability assays in Caco-2 and MDCK-MDR1 (Papp A-B / B-A results 1.8 / 4 and 5 / 5 10−6 cm/s); (iv) inhibits cancer cell proliferation in a panel of 17 cancer cell lines; and (v) suppresses glucose uptake in cancer cells. Rodent PK studies following IV dosing at 5 mg/kg resulted in a profile with a satisfactory half-life (5.1 hours), exposure (AUCinf 1804 ng.h/ml), tissue distribution (Vd 20.5 L/kg) and reasonable clearance (46.2 mL/min/kg). Also, pre-clinical efficacy studies of C57Bl/6 mice bearing Lewis Lung Carcinoma (LLC) xenografts demonstrated ∼80% tumor growth inhibition relative to vehicle control. Finally, micro-PET studies performed on mice bearing LLC tumors showed a significant inhibition of tumor 2-[18F]-fluoro-2-deoxy-glucose uptake. These studies demonstrate that PFK-015 is an effective inhibitor of PFKFB3, possesses compelling in vitro properties, has satisfactory PK properties in rodents, and suppresses tumor glucose metabolism and growth in an aggressive mouse model of non-small cell lung cancer. These results support further development of PFK-015 as a novel anti-cancer agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2825. doi:10.1158/1538-7445.AM2011-2825

Parallel activation of mitochondrial oxidative metabolism with increased cardiac energy expenditure is not dependent on fatty acid oxidation in pigs

Steady state concentrations of ATP and ADP in vivo are similar at low and high cardiac workloads; however, the mechanisms that regulate the activation of substrate metabolism and oxidative phosphorylation that supports this stability are poorly understood. We tested the hypotheses that (1) there is parallel activation of mitochondrial and cytosolic dehydrogenases in the transition from low to high workload, which increases NADH/NAD+ ratio in both compartments, and (2) this response does not require an increase in fatty acid oxidation (FAO). Anaesthetized pigs were subjected to either sham treatment, or an abrupt increase in cardiac workload for 5 min with dobutamine infusion and aortic constriction. Myocardial oxygen consumption and FAO were increased 3- and 2-fold, respectively, but ATP and ADP concentrations did not change. NADH-generating pathways were rapidly activated in both the cytosol and mitochondria, as seen in a 40% depletion in glycogen stores, a 3.6-fold activation of pyruvate dehydrogenase, and a 50% increase in tissue NADH/NAD+. Simulations from a multicompartmental computational model of cardiac energy metabolism predicted that parallel activation of glycolysis and mitochondrial metabolism results in an increase in the NADH/NAD+ ratio in both cytosol and mitochondria. FAO was blocked by 75% in a third group of pigs, and a similar increase in and the NAHD/NAD+ ratio was observed. In conclusion, in the transition to a high cardiac workload there is rapid parallel activation of substrate oxidation that results in an increase in the NADH/NAD+ ratio.

Systems of biochemical reactions from the point of view of a semigrand partition function

Semigrand partition functions contain all the thermodynamic information on reaction systems. When they are written for systems at specified pH, they yield the transformed Gibbs energy G′ of the system and the thermodynamic properties that can be calculated from G′. When they are written for systems at specified pH and specified concentrations of coenzymes, they yield the further transformed Gibbs energy G″ and properties that can be calculated from G″. This is illustrated by considering: (1) a reactant that is a weak monoprotic acid at a specified pH; (2) a reaction between two pseudoisomer groups at a specified pH; and (3) the first five reactions of glycolysis. Equilibrium compositions in glycolysis are calculated at pH 7 and different steady-state concentrations of ATP and ADP.

Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations.

The in vivo activity of nitrogenase under aerobiosis was studied with diazotrophic chemostat cultures of Azotobacter vinelandii grown under glucose- or phosphate-limited conditions at different dilution rates (Ds, representing the growth rate mu) and different dissolved oxygen concentrations. Under steady-state conditions, the concentration as well as the cellular level of ATP increased in glucose-limited cultures when D was increased. Irrespective of the type of growth limitation or the dissolved oxygen concentration, the steady-state concentrations of ATP and of dinitrogen fixed by nitrogenase increased in direct proportion to each other. Specific rates of dinitrogen fixation as well as of the regeneration of the cellular ATP pool were compared with specific rates of cellular respiration. With glucose-limited cultures, the rate of regeneration of the ATP pool and the rate of respiration varied in direct proportion to each other. This relationship, however, was dependent on the dissolved oxygen concentration. As compared to the phosphate-sufficient control, phosphate-limited cultures exhibited the same nitrogenase activity but significantly increased respiratory activities. Rates of ATP regeneration and of cellular respiration of phosphate-limited cultures did not fit into the relationship characteristic of glucose-limited cultures. However, a linear relationship between the rates of dinitrogen fixation and ATP regeneration was identified irrespective of the type of growth limitation and the dissolved oxygen concentration. The results suggest that the ATP supply rather than cellular oxygen consumption is of primary importance in keeping nitrogenase activity in aerobic cultures of A. vinelandii.

Localized firefly luciferase probes ATP at the surface of mitochondria.

The concentration of ATP generated by yeast mitochondria and consumed by yeast hexokinase was monitored using native firefly luciferase in solution, or recombinant luciferase localized at the surface of mitochondria. In the absence of hexokinase, both probes perform similarly in detecting exogenous or mitochondrially-generated ATP. The steady-state concentrations of ATP can be reduced in a dose-dependent manner by hexokinase. With hexokinase added in large excess, the localized probe reports substantial ATP concentrations while none is detectable by soluble luciferase. Thus, ATP accumulates near the membrane where it appears, relatively to solution, and vice versa for ADP. The extent of nucleotide gradients is shown to be correlated with the specific activity of oxidative phosphorylation and with the viscosity of the medium, but independent of the concentration of the organelles. A simple model involving diffusional restrictions is presented to describe this behavior. The metabolic and evolutionary implications of cellular catalysis limitation by physical processes are discussed.

Stimulation of mitochondrial oxygen consumption in isolated cardiomyocytes after hypoxia-reoxygenation.

An increase in mitochondrial matrix free calcium has been shown to occur during oxygen and substrate deprivation of the perfused heart which predisposes to calcium overload and inhibition of mitochondrial function on reoxygenation. In the current study we have assessed the effect of substrate free hypoxia on mitochondrial oxygen consumption and ATP synthesis in isolated rat cardiomyocytes. Myocytes were subjected to 40 min of substrate-free hypoxia and the oxygen consumption measured together with the effects on ATP and PCr synthesis. After hypoxia myocytes showed a fall in ATP to 10% of the control value. Within 5 sec of reoxygenation the ATP level recovered to a new steady state level of 30% of the original value. The rate of oxygen consumption of hypoxic/reoxygenated cells was 3-4 fold higher than that of cells maintained under normoxic controls but in the presence of oligomycin the difference was only 1.5-fold higher, indicating a greater requirement for mitochondrial synthesis of ATP following reoxygenation. Reoxygenation in the absence of extracellular Ca2+ resulted in a lower rate of oxygen consumption (50% of the rate measured in the presence of 1 mM-Ca2+) but did not affect the steady state concentration of ATP attained 5 min after reoxygenation. These results support the idea that the increased O2 consumption of myocytes following hypoxia/reoxygenation is due to an increased demand for ATP synthesis by mitochondria and is a response to the NA+ and Ca2+ loading of the cells which occurs under these conditions. This increased demand is likely to result in a greater generation of free radicals such as superoxide by the respiratory chain which could impair cellular function over the long term.

EFFECTS OF LOW O2 ON SENESCENCE OF CARNATION FLOWERS (DIANTHUS CARYOPHYLLUS L. cv. ELLIOTT'S WHITE)

Additions of adjuvants which inhibit the biosynthesis or action of C2H4 show that the climacteric rise in respiration during senescence of cut carnations is a facet of ethylene action and not senescence as such. The rate of CO2 output of carnation flowers was diminished in a dose-dependent mode by low O2. The data indicate that the diminution of respiration by low O2 may not be attributed to the restriction of either of the mitochondrial terminal oxidases. The steady-state concentration of ATP was similar in both air and 2% O2-treated flowers. 2% O2 eliminated for 32 days any rise in C2H4 evolution. In addition the longevity of the flowers kept under 2% O2 was longer than those which were treated with STS. The results are taken to indicate that hypoxia affects developmental events leading to the induction of C2H4 and/or the synthesis of transducer of C2H4 action.

Kinetic alterations of cytochrome c oxidase in carbon tetrachloride induced cirrhotic rat liver

In a study of the chronic effects of CCl 4 on the respiratory activities of rat liver mitochondria, the content of cytochrome c oxidase increased from 0.077±0.010 (nmol/mg protein) for normal rats to 0.101±0.009, and its specific activity increased from Vmax=345±24 (e −/s/cytochrome aa 3) to 431±19 in mitochondria of CCl 4 treated rats. There was a slight increase in Km for cytochrome c from 5.63±0.08 μM to 7.79±0.80. These results would strongly suggest that an appreciable decrease in the steady state concentration of ATP in hepatic cells of CCl 4 treated rats brought about a compensatory increase in the overall activity of cytochrome c oxidase. However, when the rate of oxygen uptake by mitochondria was measured in the presence of rotenone and tetramethyl-p-phenylene-diamine with NADH as substrate, the specific activity in CCl 4 treated rats was lower than that of normal rats (Vmax=345±31 (e −/s/cytochrome aa 3), as compared to Vmax=408±21) in spite of the increased activity of cytochrome c oxidase. This phenomenon was ascribed to a decrease in the activity of NADH cytochrome b 5 reductase in the mitochondrial outer membrane due to CCl 4 treatment.

Comparison of the effect of 3- and 5-day hypothermic perfusion of dog kidneys on metabolism of tissue slices

Hypothermic perfusion effectively preserves the viability of kidneys for 3 days. Long-term preservation (5 days or greater) has not been consistently obtained. In this study, the differences between kidneys perfused for 3 and 5 days were compared by determining the “integrated-metabolic” capabilities of tissue slices incubated in vitro at 30 °C. The “integrated-metabolic” parameters determined include (1) respiration rates, (2) cell volume regulation [total tissue water (TTW) and saccharide permeable space], (3) rate of reaccumulation of K + and pumping of Na +, (4) maintenance of ATP concentrations, and (5) mitochondrial functions. Conditions that result in high and low concentrations of ATP following perfusion of kidneys for 5 days were also compared for effects on tissue slice metabolism. The results indicate that energy metabolism in tissue slices is well preserved under all conditions and times of perfusion of kidneys. This includes average respiration rates (315 ± 50, 275 ± 35, and 255 ± 45 μmol O 2/hr/g dry wt at 0, 3, and 5 days, respectively, mitochondrial function [respiratory control ratio (RCR) = 4.6, 4.0, and 4.1 for 0, 3, and 5 days, respectively], and steadystate concentration of ATP in slices after incubation (4.0 ± 1.45, 3.9 ± 1.28, and 3.3 ± 0.81 μmol/g/ dry wt, for 0, 3, and 5 days, respectively). The primary differences between 3- and 5-day perfused kidneys were the capability of the slices to regulate cell volume and reaccumulate K +. Slices from kidneys perfused for 3 days maintained the TTW at 3.8 kg/kg dry wt, a value similar to that of control tissue slices. However, slices from 5-day perfused kidneys remained swollen (TTW = 4.6 kg/kg dry wt). Also, slices from the 5-day perfused kidney pumped K + at less than one-half the rate found in slices from control or 3-day preserved kidneys. No significant differences were apparent in the permeability properties of the tissue slices from kidneys perfused for 3 and 5 days to radiolabeled saccharides. The defects in membrane-linked transport functions, resulting from long-term kidney perfusion, were reduced in kidneys containing a high concentration of ATP. The results suggest that one factor which may limit successful preservation of kidneys is the increased membrane permeability (to electrolytes) which is partially prevented by maintaining elevated concentrations of tissue ATP during perfusion.

Gradients of O2 concentration in hepatocytes.

1. Cytochrome P/450-dependent mixed function oxidations of hexobarbital, phenyramidol, and alprenolol in intact hepatocytes were examined at different steady state oxygen concentrations. Apparent Kmo2 values were determined to be 6.4 +/- 1.7, 3.6 +/- 0.6, and 9.8 +/- 1.2 micronM, respectively. 2. Apparent Kmo2 values for metabolism of hexobarbital and alprenolol by liver microsomes were 4.3 +/- 0.4 and 8.7 +/- 0.7 micronM, similar to the corresponding values for whole cells. Therefore, no detectable gradient of O2 concentration exists between extracellular space and endoplasmic reticulum of hepatocytes at these oxygen concentrations. 3. Steady state concentrations of ATP, ADP, AMP, lactate, and pyruvate at different steady state oxygen concentrations were used as indicators of mitochondrial oxygen dependence in intact hepatocytes. Half-maximal changes occurred at [O2] = 12.6 micronM for cytoplasmic [NAD+]/[NADH] (estimated from [lactate]/[pyruvate]), at 7.0 micronM for [ATP]/[ADP], and at 2.8 micronM for adenylate energy charge. The apparent cellular respiratory Kmo2 was 1.90 +/- 0.18 micronM. 4. Comparison of values for oxygen dependence of mitochondrial functions in isolated hepatocytes with published values for isolated mitochondria suggests that a substantial intracellular oxygen gradient exists between the outer cellular membrane and the mitochondrial inner membrane at po2 values below the critical O2 tensions.

Studies on the Action of Insulin in Isolated Adipose Tissue Cells: I. STIMULATION OF INCORPORATION OF 32P-LABELED INORGANIC PHOSPHATE INTO MONONUCLEOTIDES IN THE ABSENCE OF GLUCOSE

Abstract 1. Insulin stimulated the incorporation of 32P-labeled inorganic phosphate into acid-soluble mononucleotides in isolated fat cells incubated in the absence of glucose in the medium. The specific radioactivity of the ATP increased by approximately 60%. 2. This increase was accompanied by a small but significant increment in steady state concentration of ATP over control. 3. Experiments with previously labeled cells, incubated in the absence of phosphate in the incubation medium, suggest that this stimulation by insulin of mononucleotide labeling was not a result of an effect of insulin on active transport across the plasma membrane. 4. Lipolytic agents, which are known to increase the concentration of cyclic AMP in adipose cells, diminished the incorporation of 32Pi into monucleotides. They also decreased ATP concentration, suggesting that fatty acids, their derivatives, or an unknown factor connected with lipolysis impaired oxidative phosphorylation. Insulin diminished the inhibitory action of lipolytic agents and restored ATP concentration.

AEROBIC AND ANAEROBIC P32LABELLING OF PHOSPHOLIPIDS AND ADENOSINE PHOSPHATES IN HYPOTONIC HOMOGENATES OF RAT BRAIN

Under aerobic conditions, a suitably 'reinforced' homogenate of rat brain prepared in distilled water was capable of labelling lipid P, ATP, ADP, and AMP from inorganic P32. The labelling was 'uncoupled' by the addition of DNP. The DNP decreased the specific activity of each of the above compounds and decreased the steady-state concentrations of ATP, with an increase in the concentrations of inorganic P, ADP, and AMP. Fluoride increased the specific activity of the lipid P and the steady-state concentration of ATP. Cyanide decreased the specific activity of lipid P and both the specific activity and the steady-state concentration of ATP.Under anaerobic conditions, the labelling of the lipid P and the adenosine phosphates was as good as that observed in the presence of oxygen. The anaerobic labelling of both lipid P and the adenosine phosphates was less sensitive to DNP or cyanide and more sensitive to iodoacetate than the aerobic labelling. In contrast to the aerobic labelling, the anaerobic labelling was inhibited by fluoride.In most instances, changes in the labelling of lipid P corresponded to those observed for the labelling of ATP. An exception was the labelling of lipid P that occurred under aerobic conditions in the presence of fluoride. Here the specific activity of the lipid P was related to the concentration rather than to the specific activity of ATP.