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PubMed2003

Sarcolemmal and mitochondrial effects of a KATP opener, P-1075, in "polarized" and "depolarized" Langendorff-perfused rat hearts.

We investigated consequences of cardiac arrest on sarcolemmal and mitochondrial effects of ATP-sensitive potassium channel (KATP) opener, P-1075, in Langendorff-perfused rat hearts. Depolarised cardiac arrest (24.7 mM KCl) did not affect glibenclamide-sensitive twofold activation of rubidium efflux by P-1075 (5 microM) from rubidium-loaded hearts, but eliminated uncoupling produced by P-1075 in beating hearts: 40% depletion of phosphocreatine and ATP, 50% increase in oxygen consumption, and reduction of cytochrome c oxidase. Depolarized cardiac arrest by calcium channel blocker, verapamil (5 microM), also prevented uncoupling. Lack of P-1075 mitochondrial effects in depolarized hearts was not due to changes in phosphorylation potential, because 2,4-dintrophenol (10 microM) reversed the [PCr]/[Cr] increase and Pi decrease, characteristic of KCl-arrest, but did not restore uncoupling. In agreement with this conclusion, pyruvate (5 mM) increased [PCr]/[Cr] and decreased Pi, but did not prevent uncoupling in beating hearts. A decrease in mean [Ca2+] in KCl-arrested hearts could not account for lack of P-1075 mitochondrial effects, because calcium channel opener, S-(-)-Bay K8644 (50 nM), and beta-agonist, isoproterenol (0.5 microM), did not facilitate uncoupling. In contrast, in adenosine (1 mM)-arrested hearts (polarized arrest), P-1075 caused 40% phosphocreatine and ATP depletion. In isolated rat liver mitochondria, P-1075 (20 microM) decreased mitochondrial membrane potential (DeltaPsi) by approximately 14 mV (demonstrated by redistribution of DeltaPsi-sensitive dye, rhodamine 800) in a glibenclamide-sensitive manner. We concluded that cell membrane depolarization does not prevent activation of sarcolemmal KATP by P-1075, but it plays a role in mitochondrial uncoupling effects of P-1075.

Animals↗
PubMed2005

Creatine supplementation normalizes mutagenesis of mitochondrial DNA as well as functional consequences.

Mutations of mitochondrial (mt) DNA play a role in neurodegeneration, normal aging, premature aging of the skin (photoaging), and tumors. We and others could demonstrate that mtDNA mutations can be induced in skin cells in vitro and in normal human skin in vivo by repetitive, sublethal ultraviolet (UV)-A-irradiation. These mutations are mediated by singlet oxygen and persist in human skin as long-term biomarkers of UV exposure. Although mtDNA exclusively encodes for the respiratory chain, involvement of the energy metabolism in mtDNA mutagenesis and a protective role of the energy precursor creatine have thus far not been shown. We assessed the amount of a marker mutation of mtDNA, the so-called common deletion, by real-time PCR. Induction of the common deletion was paralleled by a measurable decrease of oxygen consumption, mitochondrial membrane potential, and ATP content, as well as an increase of matrix metalloproteinase-1. Mitochondrial mutagenesis as well as functional consequences could be normalized by increasing intracellular creatine levels. These data indicate that increase of the energy precursor creatine protects from functionally relevant, aging-associated mutations of mitochondrial DNA.

Adenosine Triphosphate↗
PubMed1996

Energy metabolism of adriamycin-sensitive and -resistant Ehrlich ascites tumor cells.

Respiration, glycolysis, utilization of carbon from 14C-labeled glucose and the activities of some regulatory enzymes of the Krebs cycle and glycolysis in adriamycin-sensitive (EH-WT) and -resistant (EH-ADR) Ehrlich ascites tumor cells have been investigated. The following summarizes the results: 1. Compared with wild-type cells, EH-ADR cells exhibited an enhanced rate of oxygen consumption as well as of ATP production (2-fold). 2. When the cells were supplied with glucose as the only added energy source, the aerobic lactate production was 30% higher in EH-ADR cells. However, in spite of the enhanced glycolysis, 50% of total cell ATP was still supplied by oxidative phosphorylation, whereas in EH-WT cells 65% of ATP was derived from glycolysis. 3. The activities of the regulatory enzymes were remarkably more elevated in EH-ADR cells. 4. The amount of glucose carbon atoms metabolized through the Krebs cycle and pentose phosphate pathway in EH-ADR cells was significantly higher than in EH-WT cells. 5. These differences confirmed a modified energy metabolism in resistant cells and reflected metabolic adaptations associated with the development of multidrug resistance.

Animals↗
PubMed2000

Bioenergetic response of isolated nerve terminals of rat brain to osmotic swelling.

The swelling of nerve terminals of rat brain in a hypotonic medium (230 mOsm) induced the potential-independent entrance of 45Ca2+ into synaptosomes and intrasynaptosomal mitochondria that changed the energy status of synaptosomes, the rate of O2 consumption and the content of ATP being decreased. The ratio ATP/ADP decreased from 6.5 +/- 0.26 (310 mOsm medium) to 3.1 +/- 0.18 (the medium 230 mOsm). Studies on the equilibrium distribution of K+ (86Rb+) and [3H]TPP+ showed that contents of these cations in the nerve terminals were virtually the same on incubation in both iso- and hypotonic media. This indicated that the swelling did not damage intrasynaptosomal mitochondria and plasma membranes of the synaptosomes. The inhibition of oxidative phosphorylation increased twofold the rate of glycolysis. The incubation of synaptosomes in calcium-free medium (230 mOsm) in the presence of EGTA (1 mM) prevented the inhibition of oxidative phosphorylation and synthesis of ATP by the osmotic swelling. Ruthenium Red (10 microM) in the medium 230 mOsm inhibited the entrance of 45Ca2+ into the intrasynaptosomal mitochondria and normalized the oxidative phosphorylation to the control level (310 mOsm medium). The decrease in the energy potential of synaptosomes induced by the hypoosmotic shock is suggested to be associated with the increase in Ca2+ content in the cytoplasm, its transport into the mitochondria, and the inhibitory effect on oxidative phosphorylation.

Adenosine Diphosphate↗
PubMed1984

Relationships between aerobic and anaerobic energy production in turtle brain in situ.

To define the relationships between brain mitochondrial activity and high-energy intermediates in the turtle, brains were frozen in situ at times determined by the reduction-oxidation status of cytochrome a,a3. Increases in brain lactate indicated that transition to anaerobiosis occurred while oxidative reactions were still ongoing during hypoxia and that the signal for anaerobiosis is not the complete failure of mitochondrial electron transfer. Decreases in creatine phosphate (CrP), ATP, ADP, and AMP demonstrate that their consumption exceeded production during hypoxia. CrP decreased further during the following 120 min of anoxia, but ATP, ADP, and AMP were not significantly altered from control values. These data suggest that ATP is not inhibitory to rate-limiting, key glycolytic enzymes such as phosphofructokinase. Calculations indicate that ATP production declined during anoxia. This study indicates that decreased CrP values, decreased ATP production, and high lactate levels do not preclude ion homeostasis and electrical activity of turtle brain and that this brain is protected against lactate or pH damage during anoxia, in contrast to mammalian brain.

Adenine Nucleotides↗
PubMed2004

[Oxygen uptake and maximal oxygen uptake: interests and limits of their measurements].

The paper presents a review of the interests and limits of oxygen uptake measurement in the functional testing of athletes and disabled people. The validity of the oxygen uptake as an estimation of the oxygen consumption and aerobic synthesis of ATP is discussed in the introduction of the review. The author discusses the interests of oxygen uptake measurements for the study of energy cost in addition to maximal oxygen uptake. The limits of the study of oxygen uptake kinetics at the beginning of exercise are discussed. The methodology of oxygen measurement is mainly focused on the characteristics of the different ergometers and the choice of an exercise protocol. The review ends with short statements related to the current knowledge on maximal oxygen uptake, its limiting factors and the effects of age, gender, body mass, active muscle mass and training.

Adenosine Triphosphate↗
PubMed1984

The effects of metformin compared to the effects of phenformin on the lactate production and the metabolism of isolated parenchymal rat liver cell.

The metabolic effects of metformin were compared to the effects of phenformin on isolated parenchymal liver cells from fed and fasted rats with ethanol or glycerol as the only substrate. Both biguanides caused a fall in the hepatic oxygen-consumption, in the cellular ATP-content and in the ATP/ADP-ratio. The lactate production and the concentration-ratios of lactate/pyruvate and of beta-hydroxybutyrate/acetoacetate rose. The production rate of ketone bodies remained unchanged. This response was the same whether the hepatocytes were from fed or fasted rats and whether glycerol or ethanol was substrate. Only quantitative differences in the response on the biguanides were detected. The effects of the biguanides were dose dependent. Phenformin was ten times more potent than metformin. The same holds for their therapeutical potency. The findings indicate inhibition of the oxidative phosphorylation by both biguanides resulting in reduction of the cytoplasmic and the mitochondrial redox potentials causing enhanced lactate production. It is concluded that metformin excerts the same effects as phenformin on the hepatic metabolism, when the concentration ratio is about ten to one. Both biguanides give rise to elevated lactate production. This effect is highly increased when ethanol is present.

Adenosine Triphosphate↗
PubMed1986

The role of ATP-magnesium in ischemia and shock.

Although much is known about the role of Mg in cardiomyopathies of different etiology, very little is known about the changes in hepatic Mg levels following hemorrhagic shock or ischemia to the liver. Information available indicates that tissue and mitochondrial Mg levels may be altered following shock and ischemia and that such alterations may be responsible for the depressed cellular function during those conditions. MgCl2 administration following shock or ischemia was ineffective in improving tissue and mitochondrial Mg levels and cellular functions. Administration of ATP complexed with MgCl2, however, increased tissue and mitochondrial Mg levels, tissue ATP stores and cellular functions and proved beneficial for the survival of animals. ATP-MgCl2 administration also increased cardiac output while decreasing myocardial as well as total body O2 consumption. The potential mechanisms of the beneficial effects of ATP-MgCl2 are discussed. ATP-MgCl2 can be given safely to humans and it decreases myocardial O2 consumption and increases cardiac output without producing hypotension. A clinical trial of ATP-MgCl2 in patients with various adverse circulatory conditions is underway at our institution.

Adenosine Triphosphate↗
PubMed2001

The creatine kinase system is essential for optimal refill of the sarcoplasmic reticulum Ca2+ store in skeletal muscle.

Muscle function depends on an adequate ATP supply to sustain the energy consumption associated with Ca(2+) cycling and actomyosin sliding during contraction. In this regulation of energy homeostasis, the creatine kinase (CK) circuit for high energy phosphoryl transfer between ATP and phosphocreatine plays an important role. We earlier established a functional connection between the activity of the CK system and Ca(2+) homeostasis during depolarization and contractile activity of muscle. Here, we show how CK activity is coupled to the kinetics of spontaneous and electrically induced Ca(2+) transients in the sarcoplasm of myotubes. Using the UV ratiometric Ca(2+) probe Indo-1 and video-rate confocal microscopy in CK-proficient and -deficient cultured cells, we found that spontaneous and electrically induced transients were dependent on ryanodine-sensitive Ca(2+) release channels, sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps, extracellular calcium, and functional mitochondria in both cell types. However, at increasing sarcoplasmic Ca(2+) load (induced by electrical stimulation at 0.1, 1, and 10 Hz), the Ca(2+) removal rate and the amount of Ca(2+) released per transient were gradually reduced in CK-deficient (but not wild-type) myotubes. We conclude that the CK/phosphocreatine circuit is essential for efficient delivery of ATP to the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps and thereby directly influences sarcoplasmic reticulum refilling and the kinetics of the sarcoplasmic Ca(2+) signals.

Adenosine Triphosphate↗
PubMed1976

In vitro metabolism of red blood cells from newborn and adult goats.

The erythrocytes of newborn and adult goats were studied with respect to GSH stability, GSH regeneration, levels of ATP and 2, 3-DPG, glucose consumption and activities of nine different enzymes of Embden-Meyerhof and pentose phosphate pathways of glucose metabolism. Red cells from newborn goats had significantly higher levels of ATP and 2, 3-DPG. The activities of all the enzymes measured were also significantly greater in the newborn. It is suggested that newborn goats possess red blood cells that are metabolically more active than those of adult goats.

Adenosine Triphosphate↗
PubMed1995

Effects of SH-blocking compounds on the energy metabolism in isolated rat hepatocytes.

To investigate the importance of SH-groups in the energy metabolism of liver cells, isolated rat hepatocytes were exposed to various SH-blocking compounds. After 1.0 hr exposure, the cells were analyzed for the content of glycogen, lactate, pyruvate, ATP and the rate of oxygen consumption. Without affecting the cell viability, PCMB, PCMBS, mersalyl, NEM, DTP and DSF were found to decrease glycogen levels, whereas the disulphide reagent CDPS did not affect this endogenous energy reserve. Lactate and pyruvate levels were decreased by the organic mercury compounds, whereas NEM, DTP and DSF stimulated the formation of lactate, without affecting the levels of pyruvate. In both situations the oxygen consumption was slightly decreased. The FCCP uncoupled oxygen consumption was not affected. Up to the point of loss of cell viability, as measured by trypan blue exclusion and LDH-leakage, the liver cells maintained their ATP levels, during exposure to the various SH-reagents. In conclusion, the results with organic mercury compounds suggest a reaction of these agents with SH-groups in the outer membrane of cells, having an inhibiting effect on the glucose uptake. The most prominent effect of DTP, DSF and NEM was an increased lactate formation, implying an intracellular effect, most probably in the TCA-cycle.

Animals↗
PubMed1985

Alterations in motility and metabolism associated with sperm interaction with accessory sex gland fluids.

Sperm passing through the male tract interact with accessory sex gland fluids during ejaculation. Cellular metabolism is stimulated by this interaction for unknown reasons. These experiments involved calorimetric measurements [P.B. Inskeep and R.H. Hammerstedt (1983) J. Biochem. Biophys. Methods 7, 199-210] on ejaculated sperm (EJS) and cauda epididymal sperm (CES) from bulls to establish the contribution of individual pathways to total cellular ATP synthesis. Parallel incubations outside the calorimeter yielded samples for oxygen consumption measurements and for motility analysis, the major ATP-consuming reaction of sperm. Energy charge values were identical for incubations of EJS and CES with glucose, thereby establishing that the ratios of rates of ATP synthesis and degradation were equivalent for these cells under this incubation condition. The total rate of ATP synthesis was greater for EJS than for CES (5 vs 13 mumol ATP h-1/10(8) cells) with less than 2 mumol ATP h-1 for each cell type coming from degradation of endogenous reserves. Thus, ejaculation is associated with a large increase in catabolic rate that is satisfied by degradation of extracellular glucose. No difference in percentage of motile sperm was noted, but mean velocity was lower for CES (58 micron s-1) than for EJS (85 micron s-1). A difference in forward motility pattern was observed (wig-wag to flipping). We conclude from these data that interaction with accessory sex gland fluids alters ATP requiring activities of sperm, with one obvious alteration being their motility pattern. The increase in ATP requirement is satisfied by increased degradation of extracellular substrates, but not intracellular reserves, to provide sufficient ATP to satisfy cellular needs.

Adenosine Triphosphate↗
PubMed1988

[Effect of biologically active substances on lymphocyte energetics studied by using a simple luminometer-ATP meter].

A method of lymphocyte energetics investigation according to the ATP concentration in cell suspension has been described. A simple easily reproducible luminometer was applied for ATP measurement by luminescence of luciferin/luciferase system. The conditions of cell incubation were found when the changes in mitochondrial metabolic state reflected on ATP concentration. For all this rotenone (5 nM) decreases the ATP concentration heavily than inhibits the rate of oxygen consumption. Ecto-ATPases hydrolyze quickly the low concentrations of exogenous ATP. The examples given show the possibilities of this method for studying the effect of biologically active substances on cell energetics.

2,4-Dinitrophenol↗
PubMed1989

The bioenergetics of mitochondria after cryopreservation.

The functional characteristics of rat liver mitochondria after cryopreservation with and without the addition of the cryoprotectant dimethyl sulfoxide (Me2SO) were evaluated. As criteria of functional integrity, polarographic measurements of substrate-linked oxygen consumption and luminescent assay of adenosine triphosphate (ATP) synthesis were considered before and after cryopreservation. The results demonstrated that mitochondrial damage after freezing was indicated by the polarographic studies but was not evident when ATP synthesis was considered. Me2SO present during cryopreservation was partially protective for mitochondrial substrate-linked oxygen consumption; however, simple exposure to and dilution from Me2SO effected some changes in mitochondrial function.

Adenosine Triphosphate↗
PubMed1994

Cardiac high-energy phosphates adapt faster than oxygen consumption to changes in heart rate.

To investigate the dynamic control of cardiac ATP synthesis, we simultaneously determined the time course of mitochondrial oxygen consumption with the time course of changes in high-energy phosphates following steps in cardiac energy demand. Isolated isovolumically contracting rabbit hearts were perfused with Tyrode's solution at 28 degrees C (n = 7) or at 37 degrees C (n = 7). Coronary arterial and venous oxygen tensions were monitored with fast-responding oxygen electrodes. A cyclic pacing protocol in which we applied 64 step changes between two different heart rates was used. This enabled nuclear magnetic resonance measurement of the phosphate metabolites with a time resolution of approximately 2 seconds. Oxygen consumption changed after heart-rate steps with time constants of 14 +/- 1 (mean +/- SEM) seconds at 28 degrees C and 11 +/- 1 seconds at 37 degrees C, which are already corrected for diffusion and vascular transport delays. Doubling of the heart rate resulted in a significant decrease in phosphocreatine (PCr) content (11% at 28 degrees C, 8% at 37 degrees C), which was matched by an increase in inorganic phosphate (P(i)) content, although oxygen supply was shown to be nonlimiting. The time constants for the change of both P(i) and PCr content, approximately 5 seconds at 28 degrees C and 2.5 seconds at 37 degrees C, are significantly smaller than the respective time constants for oxygen consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗
PubMed1983

Metabolic cost of the stimulated beating of isolated adult rat heart cells in suspension.

Heart cells from adult rats were induced to beat in suspension by electric field stimulation. We have gained evidence that all the rod-shaped cells in suspension were indeed beating, and that the beat had dynamic characteristics similar to those of intact heart muscle contracting under zero load. The cells were undamaged in the process, as judged by maintenance of ATP levels, morphology, and ability to beat. In gaining such evidence, we also measured the metabolic cost to the cells of beating under zero load. In cells with oxidative phosphorylation inhibited by rotenone plus oligomycin (termed anaerobic), the rate of beat-dependent lactate production suggested an equivalent rate of ATP utilization of 0.126 +/- 0.013 nmol ATP/beat per mg protein (plus isoproterenol), and 0.058 +/- 0.005 nmol ATP/beat per mg protein (minus isoproterenol). In respiring cells, the rate of beat-dependent oligomycin-sensitive oxygen consumption gave an equivalent rate of ATP utilization of 0.198 +/- 0.009 nmol ATP/beat per mg protein (plus isoproterenol), and 0.126 +/- 0.013 nmol ATP/beat per mg protein (minus isoproterenol). The cells beat with the same approximate maximum velocity whether isoproterenol was present or not. We calculate that--in the case of anaerobic cells without isoproterenol--this rate of ATP utilization can account for only about a 15% degree of activation of the contractile proteins. In addition, we have found an oligomycin-insensitive beat-dependent mitochondrial respiration of 0.023 +/- 0.006 nanoatom O/beat per mg. The cause of this respiration is not known. The total rate of oxygen consumption of cells and also the rate per beat was comparable to that measured in nonworking whole hearts.

Adenosine Triphosphate↗
PubMed1996

Activity of the plasma membrane H(+)-ATPase and optimal glycolytic flux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid.

The weak acid sorbic acid transiently inhibited the growth of Saccharomyces cerevisiae in media at low pH. During a lag period, the length of which depended on the severity of this weak-acid stress, yeast cells appeared to adapt to this stress, eventually recovering and growing normally. This adaptation to weak-acid stress was not due to metabolism and removal of the sorbic acid. A pma1-205 mutant, with about half the normal membrane H+-ATPase activity, was shown to be more sensitive to sorbic acid than its parent. Sorbic acid appeared to stimulate plasma membrane H+-ATPase activity in both PMA1 and pma1-205. Consistent with this, cellular ATP levels showed drastic reductions, the extent of which depended on the severity of weak-acid stress. The weak acid did not appear to affect the synthesis of ATP because CO2 production and O2 consumption were not affected significantly in PMA1 and pma1-205 cells. However, a glycolytic mutant, with about one-third the normal pyruvate kinase and phosphofructokinase activity and hence a reduced capacity to generate ATP, was more sensitive to sorbic acid than its isogenic parent. These data are consistent with the idea that adaptation by yeast cells to sorbic acid is dependent on (i) the restoration of internal pH via the export of protons by the membrane H+-ATPase in an energy-demanding process and (ii) the generation of sufficient ATP to drive this process and still allow growth.

Adaptation, Physiological↗
PubMed2005

How to investigate oxygen supply, uptake, and utilization simultaneously by interleaved NMR imaging and spectroscopy of the skeletal muscle.

Human skeletal muscle perfusion, oxygenation, and high-energy phosphate distribution were measured simultaneously by interleaved (1)H and (31)P NMR spectroscopy and (1)H NMR imaging in vivo. From these parameters, arterial oxygen supply (DO(2)), muscle reoxygenation rate, mitochondrial ATP production, and O(2) consumption (VO(2)) were deduced at the recovery phase of a short ischemic exercise bout. In addition, by using a reformulation of the mass conservation law, muscle maximum O(2) extraction was calculated from these parameters.

Adult↗