Reduced ATP Content Research Articles

Effect of leukotrienes on isolated vital cardiac cells--an in vitro model of cardiocytotoxic mediators in inflammation?

Leukotriene cardiotoxicity was investigated in isolated vital rat cardiocytes using leukotrienes C4, D4 and B4. Vitality after various periods of incubation was assessed microscopically and ATP content of the cardiocytes was assayed using bioluminescence analysis. Leukotrienes C4 and D4 produced no significant cardiotoxic effect whereas leukotriene B4 showed a 15% reduction of vitality and 20% reduction of ATP content after 1 hour incubation. A clear dose dependence could not be established. These results are discussed in relation to known effects of leukotrienes and it is suggested that leukotriene B4 may itself be involved directly in the inflammatory process.

Modulation of messenger RNA metabolism in experimental methyl mercury neurotoxicity.

We have investigated the effects of methyl mercury of mRNA metabolism in mouse brain cells in vivo. It was demonstrated that methyl mercury substantially reduces the rate of synthesis of ATP and poly(A)-segments of mRNAs. The molecular sizes of poly(A)-segments isolated from hnRNA and polysomal mRNA of the experimental animal brain are smaller than the dimensions of the same segments from the cellular RNA of intact mice. A fall in the mRNA polyadenylation rate seen under methyl mercury directly correlates with reduced metabolic stability (t 1/2) of the respective poly(A)+mRNA. The mean time of nuclear-cytoplasmic transport of these mRNAs (t0 is substantially increased under methyl mercury. At the same time, methyl mercury has no effect on the metabolism of brain polysomal poly(A)-mRNA. Direct addition of methyl mercury to an in vitro system containing excess ATP failed to affect the activity of poly(A) polymerase isolated from the brain of intact mice. The poison-induced alterations in the poly(A)+mRNA metabolism bring about a considerable reduction of the poly(A)+-fraction's share in the total polysomal mRNA and dramatic fall in the intracellular polysomes concentration. All the alterations in the examined metabolic parameters well correlate both with a reduced ATP content in the brain tissue and decreased rate of total protein synthesis in brain cells. Proceeding from these results as well as data from the literature, we developed a hypothetical model of a general molecular mechanism whereby methyl mercury inhibits protein synthesis in the brain.

Induction of hyperphenylalaninemia in mice by ethionine and phenylalanine.

Female NMRI mice were fed diets containing l-ethionine (0.1 and 0.3% w/w) and phenylalanine (3% w/w), as well as respective control diets. Ethionine, the S-ethylated analog of methionine, was shown to inhibit phenylalanine hydroxylase in vivo, whereby in vitro kinetics remained unaffected. Treatment with ethionine resulted in fatty liver, reduced ATP content of liver, and alterations in serum amino acid concentrations. In the high dosage ethionine group, for instance, concentrations of Ala, Gly, Ser, Met, and Phe were increased whereas concentrations of Lys, Asp, and Pro were decreased. Applying ethionine together with phenylalanine resulted in hyperphenylalaninemia and phenylketonuria. Feeding phenylalanine alone also led to decreased activity of phenylalanine hydroxylase and increased concentration of Phe in serum. Ethionine only had a minimal effect on body weight gain; however, the hyperphenylalaninemic condition induced by application of the high dosage of ethionine and phenylalanine induced severe loss of body weight. A disturbed protein synthesis and protein phosphorylation might be the underlying mechanism of ethionine-induced suppression of phenylalanine hydroxylase.

Effect of Haloxyfop and Haloxyfop-Methyl on Elongation and Respiration of Corn (Zea mays) and Soybean (Glycine max) Roots

The effects of haloxyfop {2-[4-[[3-chloro-5(trifluoromethyl)-2-pyridinyl] oxy] phenoxy] propanoic acid} and haloxyfop-methyl on elongation and respiration of primary roots of corn [Zea maysL. ‘B37 X Oh43’] and soybean [Glycine max(L.) Merr. ‘Hodgson 78’] were examined. Intact roots of etiolated seedlings were exposed to both forms of the herbicide. At a concentration of 10-6M, neither form of the herbicide had an effect on elongation or respiration of soybean roots. In contrast, elongation of corn roots was completely inhibited within 24 h of exposure to 10-6M haloxyfop. This treatment reduced the respiration rate of corn root apices by 30% but had little effect on ATP content. After 72 h of exposure to haloxyfop [10-6M], both ATP content and respiration rate of corn root apices had declined 35%. Haloxyfop-methyl produced equivalent effects on elongation and respiration of corn roots. Studies with isolated corn root mitochondria indicated that haloxyfop inhibits electron transport at relatively high concentrations (I50= approximately 2 mM for various substrates). Because significant reductions in ATP content and respiration rate of corn root apices were not detected during the period when elongation was inhibited (initial 24 h of exposure), it is concluded that impairment of respiration is a secondary response to the herbicide.

Effects of probes of membrane potential on metabolism in synaptosomes.

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

DUCHENNE MUSCULAR DYSTROPHY: NORMAL ATP TURNOVER IN CULTURED CELLS: 65

A reduction of ATP content has been observed in muscle from patients with Duchenne muscular dystrophy. We tested the hypothesis that there is an accelerated rate of ATP turnover in the cultured muscle cells and fibroblasts from patients with Duchenne muscular dystrophy. ATP and ADP levels were similar in both muscle cells and fibroblasts from normal subjects and Duchenne patients. The rates of ATP and ADP formed from radioactive adenine and the enrichment of these pools was the same. ATP degradation was evaluated during a 10-minute incubation in 5.5 mM 2-deoxyglucose or during a 72-hour period of growth. There was significantly less radioactively-labelled ATP after incubation with deoxyglucose in Duchenne cells than in normal muscle cells, but there was no difference in total ATP concentration or ADP concentration or radioactive labelling. There was no difference in ATP or ADP degradation in fibroblasts. In longterm study of fibroblasts, radioactively labelled ATP decreased to 50% of control values at 24 hours and about 30% of control values at 72 hours, but there was no difference between normal and Duchenne cells. The data suggest that ATP turnover is normal in cells from Duchenne muscular dystrophy under the study conditions.

Effect of metabolic depletion on the furosemide-sensitive Na and K fluxes in human red cells.

We report in this paper the effect of metabolic depletion on several modes of furosemide-sensitive (FS) Na and K transport in human red blood cells. The reduction of ATP content below 100 mumol/liter cells produced a marked decrease in the maximal activation (Vmax) of the outward, FS transport of Na and K into choline medium in the presence of ouabain (0.1 mM) and 1 mM MgCl2. The K0.5 for internal Na to activate the FS Na efflux was not altered by metabolic depletion. However, metabolic depletion markedly decreased the Ki for external K (Ko) to inhibit the FS Na efflux into choline medium (from 25 to 11 mM). Repletion of ATP content by incubation of cells in a substrate-rich medium recovered control levels of Vmax of the FS Na and K fluxes and of Ki for external K to inhibit FS Na efflux. The Vmax of FS Na and K influxes was also markedly decreased when the ATP content dropped below 100 mumol/liter cells. This was mainly due to a decrease in the inward-coupled transport of K and Na (NaO-stimulated K influx and the Ko-stimulated Na influx). The FS Ki/Ko exchange pathway of the Na-K cotransport, estimated from the FS K influx from choline-20 mM Ko medium into cells containing 22 mmol Na/liter cells, was also reduced by starvation. Starvation did not inhibit the FS Nai/Nao exchange pathway, estimated as FS Na influx from a medium containing 130 mM NaCl into cells containing 22 mmol Na/liter cells. The unidirectional FS 22Na efflux and influx were also measured in control and starved cells containing 22 mmol Na/liter cells, incubated in a Na medium (130 mM) at varying external K (0 to 20 mM). In substrate-fed cells, incubated in the absence of external K, FS Na efflux was larger than Na influx. This FS net Na extrusion (400 to 500 mumol/liter cells X hr) decreased when external K was increased, approaching zero around 15 mM Ko. In starved cells the net Na extrusion was markedly decreased and it approached zero at lower Ko than in substrate-fed cells. Our results indicate that the FS Na and K fluxes, and their major component, the gradient driven Na-K-Cl cotransport system, are dependent on the metabolic integrity of the cells.

Halothane Effects on ATP Content and Uridine Uptake and Phosphorylation in Tetrahymena pyriformis

The effects of halothane on uptake and phosphorylation of uridine, and on cellular ATP content were studied in Tetrahymena pyriformis, a ciliate protozoan. Exposure to halothane inhibited the accumulation of 14C-uridine into the following acid-soluble intracellular pools: UTP, UDP, UMP, and an unidentified compound. Halothane did not alter ATP content of intact cells. It is concluded that inhibition by halothane of uridine incorporation into RNA of T. pyriformis is due to effects on uridine uptake and/or phosphorylation and not due to inhibition of the RNA polymerase reaction or reduction of ATP content.

Effect of warfarin on ATP content, viability, glycosylation and protein synthesis in isolated rat hepatocytes.

The effect of warfarin on ATP content, viability and incorporation of labelled glucosamine and leucine into protein was studied in suspensions of rat hepatocytes. When incubated with warfarin the synthesis of total protein and glycoprotein was equally inhibited, and the viability and ATP content of the cells were also lowered. These observations suggest that warfarin is not an inhibitor of glycosylation of proteins in isolated hepatocytes, however, the presence of warfarin leads to reduced ATP content of the cells which causes a decrease in protein synthesis and viability.

Cerebral metabolic and circulatory effects of 1,1,1-trichloroethane, a neurotoxic industrial solvent. 2. Tissue concentrations of labile phosphates, glycolytic metabolites, citric acid cycle intermediates, amino acids, and cyclic nucleotides.

In order to obtain information on the mechanisms of neurotoxicity of 1,1,1-trichloroethane, rats maintained artificially ventilated on N2O:O2 (70:30) were exposed to a concentration of 1,1,1-trichloroethane of 8000 ppm, 43.7 mg L-1, that induces moderate ataxia in awake, spontaneously breathing animals. After 5 and 60 min of exposure, as well as after a 60-min recovery period following 60 min of exposure, the brain was frozen in situ and cortical tissue was assayed for phosphocreatine (PCr), + ATP, ADP, AMP, glycogen, glucose, pyruvate, lactate, citric acid cycle intermediates, associated amino acids, and cyclic nucleotides; in addition, purine nucleotides, nucleosides, and bases were assayed by HPLC techniques. Exposure of animals to 1,1,1-trichloroethane failed to alter blood glucose, lactate, and pyruvate concentrations. However, the solvent induced highly significant increases in tissue lactate and pyruvate concentrations that were also reflected in cisternal CSF. Associated with these changes were increases in all citric acid cycle intermediates except succinate, an increase in alanine concentration, and a rise in the glutamate/aspartate ratio. After 5 min, a small decrease in glycogen concentration also occurred. All these changes were reversed when the exposure was terminated. No changes were observed in tissue concentrations of purine nucleotides, nucleosides, and bases except for a small reduction of ATP concentration after 60 min of exposure, still noticeable after 60 min of recovery. Apart from a small reduction in cAMP concentration after 5 min of exposure, cyclic nucleotide concentrations did not change.

Perturbation of Chara Plasmalemma Transport Function by 2[4(2′,4′-Dichlorophenoxy)phenoxy]propionic Acid

Electrophysiological measurements on internodal cells of Chara corallina Klein ex Willd., em. R.D.W. revealed that in the presence of (2-[4-(2',4'-dichlorophenoxy)phenoxy]propionic acid) (diclofop) the membrane potential was very sensitive to the pH of the bathing medium. At pH 5.7, 100 micromolar diclofop caused a slow reduction in the electrogenic component of the membrane potential to the value of -123 +/- 5 millivolts. Membrane resistance initially decreased, recovered transiently, then stabilized at approximately 65% of the control value. At pH 7.0, the potential appeared to plateau around -200 millivolts before rapidly declining to -140 +/- 4 millivolts; removal of diclofop resulted in recovery of the electrogenic component. Diclofop reduced cytoplasmic ATP levels by 96.4% and 36.6% at pH 5.7 and 7.0, respectively. At pH 8.2, diclofop did not change the ATP concentration significantly, but induced a hyperpolarization of the membrane potential to near -250 millivolts, and also reduced or inhibited the dark-induced hyperpolarization; the light-induced depolarization was reduced to a lesser extent. DCMU applied in the light elicited the same response at the plasmalemma as placing cells in the dark. When K(+) channels were opened and cells depolarized with 10 millimolar K(+), diclofop induced a further depolarization of approximately 30 millivolts. Cells decoupled with HPO(4) (-2) were still sensitive to diclofop. Currents associated with OH(-) efflux and HCO(3) (-) influx, as measured with a vibrating probe technique, became spatially destabilized and reduced in magnitude in the presence of diclofop. After 60 minutes, most of the cell surface was engaged in a low level of OH(-) efflux activity. The results indicate that diclofop may be a proton ionophore at pH 7.0 and 5.7. At pH 8.2, diclofop may inhibit the operation of the H(+)-ATPase and OH(-) efflux systems associated with HCO(3) (-) transport by perturbing the control processes that integrate the two, without a reduction in ATP concentration.

Early Metabolic Changes in Rat Epidermis and Dermis Following Cutaneous Application of Irritant Doses of Tributyltin

Metabolic changes produced in rat skin by tributyltin have been determined with the aim of identifying some of the early events which may be associated with the irritant response of this compound. When the skin irritant tributyltin (TBT) was applied to rat dorsal skin as a single cutaneous dose of 67 nmoles of TBT/sp. cm, steady state levels of 0.44 nmoles/mg dry weight were attained in the viable epidermis by 1 hr. Calculations indicate that depletion of the applied dose and consequent reduction of the concentration in the viable epidermis occurred after 5–6 hr. This dose of TBT significantly reduced the oxygen consumption of epidermal slices by 1 hr. There were minimal pathological changes at this times. Maximum inhibition of oxygen consumption occurred by 8 hr (epidermal and dermal slices inhibited by 49% and 46% respectively) by which time focal epidermal and dermal separation had occurred and numerous inflammatory cells were present. Oxygen consumption returned to normal within 48 hr. The inhibition was accompanied by a concomitant reduction in ATP concentration in the epidermis with values reduced by 63% at 12 hr. ATP remained at this value for 20 hr, returning to normal by 48 hr. Lactate concentration in full thickness skin increased by 89% within 2 hr of application of tributyltin and remained elevated for up to 48 hr. The earliest detectable reductions in oxygen consumption and ATP concentration coincided with the time when the tributyltin content of the epidermis was maximal and preceded any necrotic changes. The recovery phase coincided with reduction of tributyltin concentration. It is concluded that the early toxic action of tributyltin in rat skin involves inhibition of oxidative metabolism. The resulting perturbations may result in cellular injury or dearth and these changes may be involved in production of the altered skin.

Glycoproteins in the Local Mechanism of Calcification

Glycoproteins in calcifiable matrices appear to play two different roles. As structural components, glycoproteins are associated with proteoglycans of which they share many properties and functions. In particular they are removed before the onset of calcification, therefore unmasking sites of calcium and phosphate deposition. As calcium and/or phosphate binding sites, glycoproteins appear to be associated with the mechanism of calcification. Glycoproteins may aid in transporting calcium and phosphate from points of initial deposition into the relatively inaccessible sites of mineralization. It is possible to assume also a nucleating effect and it cannot be excluded that Ca-binding glycoproteins are components of the "crystal ghosts." Glycoproteins also modulate calcium dependent phosphatase and ATPase activities. The recent discovery of an ATP-driven calcium pump associated with the plasma membrane of chondrocytes and the previous finding of an ATPase activity associated with the cartilage calcium-binding glycoprotein suggest that reduction in ATP content in matrix vesicles may be responsible for the initial deposits of mineral.

ATP as an indicator of toxicity

ATP concentrations were shown to be a fast and reliable indicator of toxicity. pH changes, chloroform, acetone, acrolein and mercuric chloride were tested for toxic effects. Acrolein and mercuric chloride were by far the most toxic substances tested. Chloroform at its solubility limit (approx 5 ml l −1) induced only a 40% reduction in ATP concentration after 6 h exposure. Mercuric chloride at 1 ppm caused nearly 100% loss of ATP in only 1 h. These experiments have demonstrated the utility of ATP concentrations in monitoring and projecting the future effects of pollutants. In conjunction with other methods of estimating sublethal and lethal effects of pollutants, ATP could be valuable in assessing the impact of potential pollutants on aquatic environments.

Inhibition of Glucose Catabolism in Rat, Hamster, Rhesus Monkey and Human Spermatozoa by α‐Chlorohydrin

Spermatozoa from the cauda epididymidis of rats treated with RS‐α‐chlorohydrin (10 mg/kg/day p. o. × 7) had the same ATP content as control spermatozoa immediately after collection, but were unable to metabolize glucose in vitro and so their ATP content declined more rapidly than that of control spermatozoa.The in vitro metabolism of glucose by spermatozoa from rat, hamster, rhesus monkey and human was inhibited by 80% or more after 15–30 min preincubation in the presence of RS‐α‐chlorohydrin concentrations of < 1, > 10, 5–10 and > 50 mM, respectively. Inhibition of glucose oxidation was correlated with a reduction in ATP concentration in the spermatozoa.These data support the hypothesis that α‐chlorohydrin acts as an antifer‐tility agent by inhibition of sperm glycolysis but suggest that RS‐α‐chlorohydrin would not be an effective contraceptive in man. However, the glucose metabolism of human spermatozoa was significantly inhibited by < 10 mM of the S‐enantiomer of α‐chlorohydrin.

Effects of dipyridamole in vivo on ATP and cAMP content in platelets and arterial walls and on atherosclerotic plaque formation

In rabbits receiving an atherogenic diet for 2 months, the ATP content of platelet rich plasma (PRP) and arterial tissue was significantly elevated as compared to normal rabbits. This increase in ATP levels of platelets from atherosclerotic rabbits was paralleled by higher basal as well as PGI2-induced cAMP levels. In arterial tissues, an increase was only obtained in PGI2-stimulated cAMP content. Treatment with dipyridamole (DPD) for 4 weeks resulted in a reduction of the ATP content in platelets and arterial tissue from atherosclerotic rabbits to values seen in normal animals. Again, the reduction of ATP content was reflected in a decrease of basal as well as PGI2-induced cAMP levels in platelets, whereas in arterial tissue a decrease was only obtained in PGI2-induced cAMP content. At the same time, DPD treatment enhanced atherosclerotic plaque formation in the aortic wall. The enhanced atherosclerotic plaque formation seen in DPD treated atherosclerotic rabbits may be linked to the inhibition of adenosine uptake, resulting in a decrease of the adenine nucleotide pools of arterial wa-l cells. The decrease also caused a reduction in PGI2-induced cAMP content. This effect may be linked to altered proliferative activity, since in many cell types, stimulation of cAMP levels results in reduced proliferation rates.

The role of ATP in the biogenesis of vaccinia virus mRNA in vitro

During in vitro RNA synthesis vaccinia virus rapidly depletes ATP but not GTP, CTP, or UTP. This reduction in ATP concentration results in a decreased rate of RNA synthesis. The RNA transcripts made when ATP becomes rate-limiting are large in size, 20 to 30 S, compared with the 8 to 12 S RNA transcripts synthesized under high ATP concentration. Furthermore, these large RNA transcripts are not significantly polyadenylated and remain virion-associated. When one supplements the RNA polymerase reaction with additional ATP the following observations are made: (a) The rate of RNA synthesis remains constant; (b) the RNA synthesized is of the appropriate monocistronic size, 8 to 12 S; (c) the RNA is polyadenylated efficiently; and (d) the RNA is released from the virus. Some of these effects are mimicked by dATP, but neither the αβ- nor the β,γ-phosphonic acid analogs of ATP will replace ATP in the supplementation scheme. Selection of appropriate ATP concentrations in the in vitro RNA polymerase reaction allows exclusive synthesis of high-molecular-weight RNA.

The effect of various drugs on adenosine triphosphate content and histamine release from rat peritoneal cell suspensions rich in mast cells.

1. Iopanoic acid and iophenoxic acid were potent inhibitors of compound 48/80-induced histamine release from rat peritoneal cell suspensions rich in mast cells and caused a parallel dose-related reduction in ATP content of these cells. 2. Lignocaine was less potent and ouabain and probenecid were ineffective in inhibiting histamine release and reducing cellular ATP content. 3. Various drugs can inhibit 48/80-induced histamine release from rat peritoneal cells and this activity may result from depletion of cellular ATP. It is apparent that little structural specificity is required for activity in this cell system.

Macrophage deformability and phagocytosis

The influence of several metabolic inhibitors and pharmacologic agents on macrophage deformation (induced by fluid shear stress) was examined in relationship to changes in ATP content and phagocytosis of latex beads. Two relatively specific inhibitors of glycolysis (iodoacetate [IA], and sodium fluoride [NaF]) and a sulfhydryl-binding agent (N-ethylmaleimide [NEM] markedly inhibited phagocytosis and reduced cell deformability. A microtubule-disrupting agent (vinblastine) and a highly specific inhibitor of glycolysis (2-deoxyglucose) markedly inhibited phagocytosis without influencing cell deformability. An organomercurial sulfhydryl binding agent p-chloromercuribenzene (PCMBS) and a microfilament-disrupting agent (cytochalasin B) inhibited phagocytosis and increased cell deformability. The effects of these agents on phagocytosis and cell deformability bore no consistent relationship to alterations in cellular content of ATP. The observation that 2-deoxyglucose, the most specific inhibitor of glycolysis examined, reduced ATP content to levels far lower (15 percent of control values) than those achieved by any other agent examined and inhibited phagocytosis without altering cell deformability, suggests that alterations in cell deformability induced by NaF, IA, NEM, PCMBS, and cytochalasin B are not due to inhibition of glycolysis per se, but instead result from direct or indirect effects of these agents on cell constituents, possibly contractile proteins, which are determinants of cell deformability. The finding that cytochalasin B, NEM, PCMBS, and IA interfere with phagocytosis and alter cell deformability, together with evidence that these agents interact with isolated actin and myosin, suggests that contractile proteins are important both in phagocytosis and as determinants of cell deformability. The observation that vinblastine, colchicines, and heavy water (D(2)O) did not alter cell deformability, even though vinblastine caused formation of intracellular crystals of microtubular protein, indicates that microtubules are not major determinants of cell deformability. The observations that beads adhered normally to surfaces of cytochalasin B- and of PCMBS-treated cells and that shear-stress induced deformation was increased whereas phagocytosis was markedly inhibited, suggest that deformation of cells around beads associated with ingestion depends on some form of cellular (contractile?) activity, whereas deformation of cells by fluid shear stress is a passive phenomenon.

Adenylate Metabolism of Embryonic Axes from Deteriorated Soybean Seeds

RNA and protein syntheses in axes excised from dry soybean (Glycine max L.) seeds at different levels of deterioration were assayed. Low rates of protein synthesis in slightly deteriorated seeds were not due to losses in ribosomal or soluble fraction activities. However, the lowered rates of RNA and protein syntheses of deteriorated seeds were associated with reduced ATP content of the tissues. Adenine and adenosine conversions to ATP were reduced in deteriorated axes, and these reductions were reflected in reduced incorporation of these compounds into RNA.