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PubMed1984

Accounting for the ATP-consuming processes in rabbit reticulocytes.

The report deals with a detailed balance of ATP production and consumption of the rabbit reticulocyte. The sum-total of ATP produced amounts to 135 mmol . 1-1 . h-1. About 70% of the ATP consumption has been accounted for by specific processes. The main contributing processes are the globin synthesis with about 28%, the Na+, K+-ATPase with 23% and the proteolysis with more than 15%. 30% of ATP consumption has not been accounted for. Cycloheximide (20 microM) leads to a dissociation between synthesis and degradation of proteins, which argues against any obligatory connection between these processes. More than 90% of the lysine liberated from mitochondria by proteolysis were reutilized for the globin synthesis demonstrating the high nitrogen economy of reticulocytes. Each of the ATP-consuming processes studied appears to control ATP production in an independent manner without competition with each other.

Adenosine Triphosphate↗
PubMed2002

Ethanol perfusion increases the yield of oxidative phosphorylation in isolated liver of fed rats.

The question arises as to the effect of ethanol on the actual yield of oxidative phosphorylation in the whole liver because of contradictory results reported in isolated hepatic mitochondria. The adenosine triphosphate (ATP) content of liver isolated from fed rats and perfused in the presence (10 mM) and absence of ethanol was continuously evaluated using 31P Nuclear Magnetic Resonance (NMR). An accurate estimation of mitochondrial ATP synthesis in the whole organ was obtained by subtracting the glycolytic ATP supply from the total ATP production. Simultaneously, the respiratory activity was assessed using O(2) Clark electrodes. The data indicate that ethanol enhanced the net consumption of ATP, leading to a new steady state of the ATP content. ATP synthesis was also found higher under ethanol [1.86+/-0.02 micromol/min g wet weight (min g ww)] than in control [1.44+/-0.18 micromol/min g ww]. However, mitochondrial respiration remained unchanged [2.20+/-0.13 micromol/min g ww] and, consequently, the in situ mitochondrial ATP/O ratio increased from 0.33+/-0.035 (control) to 0.42+/-0.015 (ethanol). The increase of the oxidative phosphorylation yield in the whole liver may be linked to the decrease in cytochrome oxidase activity induced by ethanol [FEBS Lett. 468 (2000) 239]. The significant raise (27%) of the ATP/O ratio was not sufficient to maintain the ATP level following ethanol-increased ATP consumption.

Adenosine Triphosphate↗
PubMedDate not supplied

Sensitization to hyperthermia by 3,3'-dipentyloxacarbocyanine iodide: a positive correlation with DNA damage and negative correlations with altered cell morphology, oxygen consumption inhibition, and reduced ATP levels.

The cyanine dye 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)) (concentrations of 0.5 microgram/ml to 5.0 micrograms/ml) was shown to be a potent sensitizer of Chinese hamster ovary (CHO) cells to hyperthermic cell killing at 43.0 degrees C or 45.5 degrees C, while exhibiting no cytotoxicity at 37.0 degrees C. Sensitization to hyperthermic cell killing was accompanied by an increase in damage to the DNA, as measured by DNA unwinding. The increased DNA damage correlated qualitatively with the enhanced heat killing induced by DiOC5(3). This correlation was better in cells heated at 43.0 degrees C than in those heated at 45.5 degrees C. DiOC5(3) is known to affect other cellular functions. It inhibits electron transport, uncouples oxidative phosphorylation, and inhibits calcium ATPases. The effects of DiOC5(3) on oxygen consumption and ATP content were therefore measured at 37.0 degrees C and at hyperthermic temperatures. The results demonstrated that inhibition of oxygen consumption and reduction of cellular ATP levels played no role in inducing heat sensitization in DiOC5(3)-treated cells, or in causing cell death in cells treated with heat alone.

Adenosine Triphosphate↗
PubMed1985

Metabolic effects of dehydroabietic acid on rainbow trout erythrocytes.

Oxygen consumption and ATP concentration were measured in rainbow trout erythrocytes incubated in a physiological saline containing 0, 5, 15, 30 or 60 mg/l dehydroabietic acid. DHAA caused a decrease in cellular ATP level and oxygen consumption at concentrations above 15 mg/l. Haemolysis increased markedly, when the cellular ATP concentration decreased below 1 mM. These data suggest that increased breakdown of red cells may be the primary reason for jaundice which is observed in resin acid-toxicated fish.

Abietanes↗
PubMed1987

Improved separation method for rat proximal and distal renal tubules.

We modified and improved enzyme digestion and density gradient separation procedures to obtain fractions of proximal and distal renal tubules with high yield and viability. Kidneys from two anesthetized adult Wistar rats were flushed with Krebs-Henseleit buffer (KHB) and then perfused in situ with recirculated KHB containing collagenase and hyaluronidase at 125 mmHg. Cortices were excised, minced, and incubated in KHB containing enzymes for 35 min at 37 degrees C. Dissociated tubules were removed at 10-min intervals, rinsed, and placed in KHB containing 10% calf serum, vitamins, and amino acids at 4 degrees C. Separation was achieved by suspending the tissue in 45% isosmotic Percoll layered over an undiluted Percoll cushion and centrifuging. Proximal tubules sedimented near the cushion. Distal segments were isolated in the uppermost bands of a second 35% Percoll separation. Viability was greater than 95% as measured by lactate dehydrogenase leakage and quantitated by oxygen consumption and ATP content. Basal oxygen consumption was greater than 33 nmol O2 X min-1 X mg protein-1 in all fractions and was stimulated by succinate and inhibited by amiloride and ouabain. Basal ATP content averaged 9.7 nmol/mg ATP. An average 3.3-fold separation for the proximal fraction and 24.5-fold separation for the distal fraction was assessed by the enrichment of six specific enzyme markers, with several of the markers indicating separations up to 32-fold. Isolated tubules also displayed functional responses to parathyroid hormone and vasopressin. Distal, but not proximal, segments demonstrated significantly increased adenosine 3',5'-cyclic monophosphate formation with vasopressin.

Animals↗
PubMed2004

Proliferative responses observed following vancomycin treatment in renal proximal tubule epithelial cells.

Vancomycin (VAN) is a glycopeptide antibiotic used to treat gram-positive infections. Nephrotoxicity is a common side effect observed with vancomycin therapy. However, the mechanism of vancomycin-induced nephrotoxicity has not been fully characterized. In this study we examined the effect of vancomycin on cellular proliferation in renal proximal tubule cells. A dose- and time-dependent increase in cell number and total cellular protein was observed following vancomycin exposure. Vancomycin exposure also caused an increase in BrdU incorporation followed by the accumulation of renal proximal tubule cells in G(2)/M phase of the cell cycle. These effects were inhibited by pretreatment with the mitogen-activated protein kinase inhibitor, PD098059, suggesting an association between the cell proliferative effect of VAN and the induction of the mitogen-activated protein kinase signaling pathway. Mitochondrial function in renal proximal tubule cells was assessed using oxygen consumption and ATP concentrations. We observed an increase in oxygen consumption and ATP concentrations following short-term exposure to vancomycin. Together, our data suggest that vancomycin treatment produces alterations in mitochondrial function that coincide with a cell proliferative response in renal proximal tubule epithelial cells.

Adenosine Triphosphate↗
PubMed1998

Nitric oxide suppression reversibly attenuates mitochondrial dysfunction and cholestasis in endotoxemic rat liver.

This study aimed to examine whether nitric oxide (NO) plays a causal role in endotoxin-induced dysfunction of biliary transport. Rats were treated with intraperitoneal injection of endotoxin (O111B4, 4 mg/kg). At 2 hours, the liver was excised and perfused ex vivo with taurocholate (TC)-containing Krebs-Ringer solution under monitoring bile output and NO2 in the perfusate and tissue cyclic guanosine monophosphate (cGMP) levels as indices of NO production. The endotoxin treatment evoked a marked decrease in the bile acid-dependent bile formation concurrent with the increasing NO2 output, cGMP elevation, and a reduction of hepatic adenosine triphosphate (ATP) contents and oxygen consumption. Perfusion with 1 mmol/L aminoguanidine (AG), an inhibitor of inducible NO synthase, but not with L-nitroarginine methyl ester, an inhibitor of the constitutive form of the enzyme, significantly reversed the endotoxin-induced increment of the bile formation in concert with the recovery of oxygen consumption and ATP levels. Laser confocal microfluorography of the liver lobules using rhodamine 123 (Rh), a fluoroprobe sensitive to mitochondrial membrane potential, revealed that endotoxin elicited a significant mitochondrial dysfunction panlobularly. The AG administration reversed the endotoxin-induced decrease in mitochondrial membrane potential. Collectively, up-regulation of NO by inducible NO synthase accounts for a mechanism through which endotoxin impairs the bile formation, and its suppression serves as a therapeutic strategy for improvement of hepatobiliary function.

Animals↗
PubMed1985

Proteolysis of mitochondria in reticulocytes during maturation is ubiquitin-dependent and is accompanied by a high rate of ATP hydrolysis.

The ATP-dependent breakdown of mitochondria-containing stroma proceeds via the ubiquitin-requiring pathway. The proteolysis is linked to a large ATP-cleaved consumption amounting to 1 ATP per peptide bond or more. Proteins of mitochondria-containing stroma are much better substrates of ATP-ubiquitin-dependent proteolysis than heat-denatured ones. Hemin suppresses both proteolysis and ATP hydrolysis.

Adenosine Triphosphate↗
PubMed1998

Age-related increase in mitochondrial proton leak and decrease in ATP turnover reactions in mouse hepatocytes.

Age-related changes in mitochondria, including decreased respiratory control ratios and altered mitochondrial inner membrane lipid composition, led us to study oxidative phosphorylation in hepatocytes from old (30 mo) and young (3 mo) male C57BL/J mice. Top-down metabolic control analysis and its extension, elasticity analysis, were used to identify changes in the control and regulation of the three blocks of reactions constituting the oxidative phosphorylation system: substrate oxidation, mitochondrial proton leak, and the ATP turnover reactions. Resting oxygen consumption of cells from old mice was 15% lower (P < 0.05) than in young cells. This is explained entirely by a decrease in oxygen consumption supporting ATP turnover reactions. At all values of mitochondrial membrane potential assessed, the proportion of total oxygen consumption used to balance the leak was greater in the old cells than in the young cells. Metabolic control coefficients indicate a shift in control over respiration and phosphorylation away from substrate oxidation toward increased control by leak and by ATP turnover reactions. Control of the actual number of ATP molecules synthesized by mitochondria for each oxygen atom consumed by the ATP turnover and leak reactions was greater in old than in young cells, showing that efficiency in older cells is more sensitive to changes in these two blocks of reactions than in young cells.

Adenosine Triphosphate↗
PubMed1996

Metabolic activities of Listeria monocytogenes in the presence of sodium propionate, acetate, lactate and citrate.

The effects of sodium propionate, acetate, lactate and citrate on cell proliferation, glucose and oxygen consumption, and ATP production in Listeria monocytogenes were investigated in growing and resting cells. Media pH was 6.7-6.8. Growth inhibition increased while glucose consumption continued in the presence of > or = 1% propionate, > or = 3% acetate and > or = 5% lactate in broth during incubation at 35 degrees C, indicating that glucose consumption was uncoupled from cell proliferation. Acetate and propionate were the most effective antilisterials, whereas citrate (5%) was only slightly inhibitory. Of the four salts, only lactate supported growth, oxygen consumption and ATP production. While concentrations of 1 and 5% propionate, acetate and citrate did not have an effect on oxygen consumption, they inhibited ATP production. ATP production in the presence of the four salts was consistently lower at pH 6.0 than at neutral pH. Lactate served as an alternative energy source for L. monocytogenes in the absence of glucose but became toxic to the organism in the presence of the carbohydrate.

Adenosine Triphosphate↗
PubMed2005

Microbiological parameters as indicators of compost maturity.

AIMS: The objectives of this study were to determine the changes of microbial properties of pig manure collected from pens with different management strategies and composted using different turning and moisture regimes; relate their association with humification parameters and compost temperature; and identify the most suitable microbial indicators of compost maturity. METHODS AND RESULTS: Six different microbial parameters, including total bacterial count, oxygen consumption rate, ATP content, dehydrogenase activity, and microbial biomass C and N, along with humification parameters [humic acid (HA), fulvic acid (FA) and HA : FA ratio] and compost temperature were monitored during composting. Significant positive correlations were found between temperature and microbial properties, including O2 consumption rate, ATP content, dehydrogenase activity, and microbial biomass N. The humification parameters also showed significant correlations with microbial properties of the manure compost. For instance, HA contents of pig manures was positively correlated with total aerobic heterotrophs, and microbial biomass N and C; and negatively correlated with O2 consumption rate, ATP content, and dehydrogenase activity. Among the six microbial parameters examined, dehydrogenase activity was the most important factor affecting compost temperature and humification parameters. Composting strategies employed in this study affected the speed of composting and time of maturation. If the moisture content is maintained weekly at 60% with a 4-day turning frequency, the pig manure will reach maturity in 56 days. CONCLUSIONS: The composting process went through predictable changes in temperature, microbial properties and chemical components despite differences in the initial pig manure and composting strategies used. Among the six microbial parameters used, dehydrogenase activity is the most suitable indicator of compost maturity. Compared with respiration rate, ATP content and microbial biomass procedures, dehydrogenase activity is the simplest, quickest, and cheapest method that can be used to monitor the stability and maturity of composts. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented here show that microbial parameters can be used in revealing differences between composts and compost maturity. The statistical relationship established between humification parameters and microbial parameters, particularly dehydrogenase activity, demonstrates that it is possible to monitor the composting process more easily and rapidly by avoiding longer and more expensive analytical procedures.

Adenosine Triphosphate↗
PubMed1998

Ischemia/reperfusion injury of the pancreas: a new animal model.

BACKGROUND: Ischemia/reperfusion is thought to play an important role in the development of postimplantation pancreatitis after pancreas transplantation and also in the transition of edematous pancreatitis into necrotizing pancreatitis. Previous studies have suggested that impairment of microcirculation and hence tissue oxygenation and energy metabolism may be critical steps in this process. MATERIALS AND METHODS: In landrace pigs vascular isolation of the pancreatic tail was performed. Morphological alterations, tissue oxygenation, and energy metabolism were assessed in response to 3 h of global warm ischemia and the following reperfusion. RESULTS: A rapid onset of morphological alterations immediately after reperfusion was noted. Oxygen consumption and ATP levels were markedly decreased, and tissue oxygenation was severely impaired especially during the first hour after reperfusion. ATP tissue levels and oxygen consumption 10 min after reperfusion correlated significantly with the morphological changes at the end of the experiment. CONCLUSION: These findings can be explained by a failure of nutritive capillary perfusion and concomitant shunt perfusion. Therefore an impaired microcirculation rather than an impaired oxygen utilization shortly after reperfusion is of major relevance in the development of the ischemia/reperfusion injury of the pancreas.

Adenine Nucleotides↗
PubMed1994

Involvement of Ca(2+)-stimulated adenosine 5'-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei.

The regulatory role of Ca(2+)-stimulated adenosine 5'-triphosphatase (Ca(2+)-ATPase) in Ca2+ transport system of rat liver nuclei was investigated. Ca2+ uptake and release were determined with a Ca2+ electrode. Ca(2+)-ATPase activity was calculated by subtracting Mg(2+)-ATPase activity from (Ca(2+)-Mg2+)-ATPase activity. The release of Ca2+ from the Ca(2+)-loaded nuclei was evoked progressively after Ca2+ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca2+ from the Ca(2+)-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca2+ uptake and induced a promotion of Ca2+ release from the Ca(2+)-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca(2+)-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca2+ uptake and release. Meanwhile, verapamil and diltiazem (10 microM), a blocker of Ca2+ channels, did not prevent the NAD+ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 microM) and arachidonic acid (10 microM)-induced increase in nuclear Ca2+ release, suggesting that Ca2+ channels do not involve on Ca2+ release from the nuclei. These results indicates that an inhibition of nuclear Ca(2+)-ATPase activity causes the decrease in nuclear Ca2+ uptake and the release of Ca2+ from the Ca(2+)-loaded nuclei. The present finding suggests that Ca(2+)-ATPase plays a critical role in the regulatory mechanism of Ca2+ uptake and release in rat liver nuclei.

Adenosine Triphosphate↗
PubMed2003

Regional loss of the mitochondrial membrane potential in the hepatocyte is rapidly followed by externalization of phosphatidylserines at that specific site during apoptosis.

The spatio-temporal relationship between a decrease in the mitochondrial membrane potential (MMP) and externalization of phosphatidylserines (PS) during induction of apoptosis was investigated in single freshly isolated hepatocytes. Apoptosis was induced in the hepatocytes in three different ways: attack by activated Natural Killer cells, exposure to ATP, or exposure to the inhibitor of protein synthesis cycloheximide. Fluorescence microscopy showed staining of externalized PS at those areas where the staining for MMP was lost whereas in other areas the mitochondria remained intact for longer periods of time, indicating coupling between local loss of MMP and local PS exposure. To discriminate whether the decrease in MMP itself or a decrease in ATP induced PS externalization, hepatocytes were treated with rotenone, which resulted in a rapid collapse of cellular ATP but left the MMP intact for a much longer period. Addition of fructose prevented the decrease of ATP to approximately 30% and also delayed the collapse of the MMP. This indicates that ATP was needed for the maintenance of the MMP probably via reverse action of the ATP synthase. In a subsequent study hepatocytes were incubated with Natural Killer cells for induction of apoptosis followed by addition of rotenone to deplete ATP. Under these conditions the PS staining co-localized with mitochondrial MMP indicating that PS externalization does not require a collapse in MMP. Moreover, exposure of PS was evenly distributed over the whole plasma membrane. In conclusion, we propose that after an apoptotic stimulus some mitochondria start to loose their MMP, which results in cessation of ATP production and perhaps even consumption of ATP. This results in an overall decrease in cellular ATP. ATP-consuming enzyme reactions most distal from still intact mitochondria will be most sensitive to such a decrease. Apparently the translocase that keeps phosphatidylserines inward-oriented is such a sensitive enzyme.

Adenosine Triphosphate↗
PubMed1988

Tubular hypermetabolism as a factor in the progression of chronic renal failure.

Thus, in summary, we hypothesize that nephron loss by a time-limited insult (eg, an acute immunological insult, surgical removal of five sixths of normal nephrons) may cause events in the tubules of the remaining nephrons that contribute substantially, though not exclusively, to the inexorable progression to ESRD. Specifically, hypermetabolism in the remaining tubules as assessed by oxygen consumption and ATP synthesis rates has been found to occur. This hypermetabolism in residual nephrons appears to be due to the known increased sodium transport per nephron, but nonsodium transport events also appear to be involved. With respect to the latter phenomenon, we propose that an increase in growth factor response per tubule, as a mechanism of renal hypertrophy, activates the DAG----protein kinase C----Na/H antiporter system. We have evidence in support of this sequence of events by the demonstration of a two-fold increase in membrane (particulate) protein kinase C within 24 hours after removal of the contralateral kidney and an increase in intracellular pH as assessed by NMR spectroscopy. Activation of the Na/H antiporter not only leads to proton extrusion and cellular alkalinization, which may activate cellular alkalinization, which may activate cellular enzymes such as phospholipases, but also increases intracellular Na concentration, thereby further increasing Na/K-ATPase, ATP use, and enhancing ATP synthesis. The increase in mitochondrial oxygen consumption, which accompanies the enhanced ATP synthesis, would be expected to be associated with increased oxygen radicle generation. If the tissue scavengers of oxygen radicles are not sufficient to scavenge the increase in oxygen radicles, then lipid peroxidation and tissue damage will occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1997

Coupling of kinesin steps to ATP hydrolysis.

A key goal in the study of the function of ATP-driven motor enzymes is to quantify the movement produced from consumption of one ATP molecule. Discrete displacements of the processive motor kinesin along a microtubule have been reported as 5 and/or 8 nm. However, analysis of nanometre-scale movements is hindered by superimposed brownian motion. Moreover, because kinesin is processive and turns over stochastically, some observed displacements must arise from summation of smaller movements that are too closely spaced in time to be resolved. To address both of these problems, we used light microscopy instrumentation with low positional drift (< 39 pms[-1]) to observe single molecules of a kinesin derivative moving slowly (approximately 2.5nm s[-1]) at very low (150nM) ATP concentration, so that ATP-induced displacements were widely spaced in time. This allowed increased time-averaging to suppress brownian noise (without application of external force), permitting objective measurement of the distribution of all observed displacement sizes. The distribution was analysed with a statistics-based method which explicitly takes into account the occurrence of unresolved movements, and determines both the underlying step size and the coupling of steps to ATP hydrolytic events. Our data support a fundamental enzymatic cycle for kinesin in which hydrolysis of a single ATP molecule is coupled to a step distance of the microtubule protofilament lattice spacing of 8.12 nm. Step distances other than 8nm are excluded, as is the coupling of each step to two or more consecutive ATP hydrolysis reactions with similar rates, or the coupling of two 8-nm steps to a single hydrolysis. The measured ratio of ATP consumption rate to stepping rate is invariant over a wide range of ATP concentration, suggesting that the 1 ATP to 8nm coupling inferred from behaviour at low ATP can be generalized to high ATP.

Adenosine Triphosphatases↗
PubMed1993

Age-related changes in cardiac mitochondrial energetics under the influence of calcium in rat.

Mitochondria extracted from the hearts of Wistar rats aged 6 and 24 months showed similar values for the respiratory control index (RCI), state 3 oxygen consumption (QO2) and ADP/O measured using glutamate or succinate as substrates; with the exception that the QO2 of the aged rats was lower than that of the young rats in the presence of glutamate. The consumption of O2 induced by 2-oxoglutarate and ADP was similar in both age groups. Concentrations of external free Ca2+ ranging from 0.2 to 0.8 microM produced an increase in O2 consumption and ATP formation in the young mitochondria, with a maximum effect at 0.2 microM external free Ca2+. Little or no change in O2 consumption and ATP formation was evident in aged mitochondria following incubations in concentrations of external free Ca2+ ranging from 0.2 to 0.8 microM. The continuous rate of formation of ATP, measured in the presence of 0.2 microM external free Ca2+ using a luminescence method, confirmed the previous results. This study indicates that the cardiac mitochondrial phosphorylating system of aged rats is poorly sensitive to variations in external free calcium.

Adenosine Triphosphate↗
PubMed2003

Differential effects of "Advanced glycation endproducts" and beta-amyloid peptide on glucose utilization and ATP levels in the neuronal cell line SH-SY5Y.

Beta-amyloid peptide (Abeta) and "Advanced glycation endproducts" (AGEs) are components of the senile plaques in Alzheimer's disease patients. It has been proposed that both AGEs and Abeta exert many of their effects, which include the upregulation of pro-inflammatory cytokines, through RAGE ("receptor for advanced glycation endproducts"). To investigate whether Abeta and AGEs cause similar or identical effects on cell survival and energy metabolism, we have compared the effects of a model-AGE and Abeta on cell viability, ATP level, glucose consumption and lactate production in the neuroblastoma cell line SH-SY5Y. The results show that AGEs and Abeta increase glucose consumption and decrease ATP levels in a dose dependent manner. Furthermore, both compounds decrease mitochondrial activity measured by the MTT assay. However, only AGEs decrease the number of cells and significantly increase lactate production. These data indicate that both AGEs and Abeta can cause differential disturbances in neuronal metabolism, which may contribute to the pathophysiological findings in Alzheimer's disease. However, their signalling pathways are apparently quite distinct, a fact which should stimulate a more detailed investigation in this field, e.g. for the purpose of a rational design of potential "neuroprotective" RAGE antagonists.

Adenosine Triphosphate↗