Decrease In ATP Content Research Articles

Adriamycin: Effect on mammalian cardiac cells in culture I. Cell population and energy metabolism

The use of adriamycin, an effective cancer chemotherapeutic agent, may be precluded because of cardiotoxic side effects of unknown etiology. Mammalian cardiac cells in culture were used as a model system in the study of the mechanisms involved. Adriamycin treatment inhibited cell growth and cytochemical staining demonstrated that the cell population was enriched in myocardial cells following the drug treatment. The fast-dividing non-myocardial cells were particularly susceptible to adriamycin. The latter might be a contributing factor in the cardiomyopathy but the cessation of the rhythmic contractions characteristic of myocardial cells in culture remains to be elucidated. The analysis of acid soluble cells extracts showed a 30% decrease in intracellular concentrations of ATP and phosphorylcreatine. The addition of creatine to adriamycin treated cells resulted in a several-fold increase in the steady state concentration of phosphorylcreatine. Therefore, the regulation of energy production and the potential to maintain a high steady state concentration of phosphorylcreatine were not impaired.

Isolation and characterization of adult rat heart cells

Adult rat heart muscle cells were isolated after simultaneous perfusion of multiple (two to eight) hearts with buffered salt solutions containing collagenase and hyaluronidase. Yields (35 to 50% of ventricular weight with approximately 70% viability) are quantitatively suitable for metabolic studies. Viability has been determined by the ability of intact cells to exclude trypan blue and the inability of intact cells to oxidize exogenous succinate. Micrographs show that the fine structure of the isolated cells is well ordered. Cell concentrations of glycogen, glucose 6-phosphate, citrate, and various enzymes were similar to those of intact heart. ATP and creatine phosphate concentrations were lower than in whole hearts. Adenosine 3′,5′-monophosphate concentrations were somewhat elevated. Deoxyribonucleic acid was lower than in whole tissue. The isolated cells retain certain metabolic control mechanisms. The uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, increased oxygen consumption severalfold, whereas exogenous ADP had no effect on respiration. Under anaerobic conditions the rates of glucose utilization and lactate production were faster than in the presence of oxygen, indicating retention of the Pasteur effect. The addition of glucose and insulin caused a decrease in oxygen uptake or the Crabtree effect. Exogenously added pyruvate decreased glycolytic flux and produced a pronounced increase in intracellular citrate and glucose 6-phosphate. Isoproterenol stimulated adenylate cyclase activity of the isolated cells at the same concentrations effective with intact heart preparations. Isoproterenol and glucagon caused the activation of phosphorylase. The cells deteriorated as a function of incubation time, as indicated by a decrease in ATP content and a loss of lactate dehydrogenase into the medium. Cell deterioration was greatly accelerated by Ca 2+ at concentrations greater than 10 −5 m.

Effects of Ethanol on Bicarbonate-Stimulated ATPase, ATP, and Cyclic AMP in Canine Gastric Mucosa

SummaryPrevious reports have demonstrated that ethanol inhibits acid secretion and active transport in the gastric mucosa. In the present studies, enzymes which have been implicated in gastric active transport (Mg2+ and Mg2+ HCO3 ATPases), as well as ATP and cAMP, were evaluated in response to ethanol. The activities of Mg2+ and Mg2+HCO3-stimulated ATPases were not altered at concentrations below 10% ethanol, but were significantly inhibited with 15 and 20%. Tissue slice incubations with 20% ethanol show significantly decreased ATP concentrations at 10, 20, and 30 min. Exposure of the dog gastric mucosa in vivo to 20% ethanol produced a significant decrease in ATP content but did not alter the cAMP level. These studies suggest that the ethanol-produced decrease in gastric ATP content may be involved in the inhibition of active ion transport observed with ethanol, but that cyclic AMP is probably not involved.

ATP metabolism in an ethanol-induced fatty liver.

Abstract A marked decrease in the hepatic concentration of ATP lowers the phosphate potential of the liver of rats fed ethanol for prolonged periods of time. Either a decreased synthesis or an increased demand could account for this change in ATP. The experiments presented here were designed to investigate both possibilities. Changes in the utilization of ATP were assessed by measuring the activity of the [Na+ + K+]-activated ATPase system, and changes in the synthesis of ATP by the activity of the adenine nucleotide translocase system. Since the level of long-chain CoA derivatives of fatty acids regulate the translocation of ADP into mitochondria, their total cellular content and mitochondrial level were also determined. The experiments were conducted on male Sprague-Dawley rats (275–300 g) maintained on a liquid diet having 36 per cent of the caloric intake as ethanol. After 2 weeks, the experimental animals had a fatty liver. This change was not associated with any significant alteration in the ATP content of the liver or in the activity of the adenine nucleotide translocase system. After 4 weeks, the excess of neutral lipid in the liver still persisted, but was associated with a marked increase in the level of long-chain CoA derivatives of fatty acids. The level of ATP in the liver was only 50 per cent of normal, and the rate of translocation of ADP into the mitochondria was decreased. The activity of the adenine nucleotide translocase system could be restored to normal values if the long-chain CoA derivatives of fatty acids were removed from the surface of the mitochondrial membrane by treatment of the preparation with defatted albumin. At this time there was only a slight (15%) enhancement of the [Na+ + K+]-activated ATPase system. With the removal of ethanol from the diet the ATP level returned to normal rather quickly. Changes in the level of hepatic ATP correlated well with the activity of the adenine nucleotide translocase system but did not parallel changes in the activity of the Na+ pump system. These findings indicate that the dominant feature leading to the decrease in the ( ATP ADP × P i ) ratio is decreased synthesis of ATP.

Effects of trypsin on protein synthesis in macrophages

Treatment of rabbit alveolar macrophages with crystalline trypsin (0.04–2 mg/10 8 cells) inhibits protein synthesis and results in increased leakage of cell proteins. Trypsinization does not significantly decrease cellular DNA content or viability, and it does not increase protein breakdown. Trypsin treatment results in decreased oxidation of [1- 14C]glucose and [6- 14C]glucose, and also a decrease in ATP content. Trypsinization also causes a depression of net leucine transport and a reduction in the translational activity of polyribosomes. When normal and trypsinized macrophages are preincubated at 37 °C for several hours and then pulse-labelled with radioactive leucine, protein synthesis is stimulated to approximately the same extent in both the control and the enzyme-treated cells. Since the trypsinized cells still exhibit depressed protein synthesis, this suggests that the inhibition cannot be readily reversed. Indirect evidence indicates that the inhibition of protein synthesis is not due to entry of trypsin into the cells and suggests that the inhibition is due to changes in metabolism resulting from the action of the enzyme at the cell surface.

Production of 1,2-diacylglycerol and phosphatidate in human erythrocytes treated with calcium ions and ionophore A23187

1. When the ionophore A23187 and Ca2+ were added to normal human erythrocytes, the incorporation of 32P into phosphatidate was enhanced within 1 min, but there was only slight labelling of other phospholipids. 2. Labelling of phosphatidate in these cells did not continue to increase after about 20min at 37 degrees C; by this time, radioactivity in phosphatidate was about ten times higher inionophore A23187-treated cells than in controls. A net synthesis of phosphatidate was measured in response to the increase in intracellular Ca2+ concentration; the content of this phospholipid in the cell was increased by approximately 50%. 3. In the presence of 2.5 mM-Ca2+ a maximum effect was seen with about 0.5 mug of ionophore/ml. 4. The concentration of Ca2+ giving half-maximal labelling of phosphatidate in the presence of 10 mug of ionophore A23187/ml was about 10 muM. 5. A rapid decrease of ATP content in the cell occurred in ionophore-treated cells. 6. Labelling of phosphatidate appeared to be secondary to the production of 1,2-diacylglycerol in the cells; accumulation of 1,2-diacylglycerol was only seen after about 15 min. After 60 min, the 1,2-diacylglycerol content of the cells was five to seven times that of untreated control cells. 7. The change in the shape of erythrocytes treated with Ca2+ and ionophore appeared to be related to accumulation of 1,2-diacylglycerol. 8. The source of 1,2-diacylglycerol has not been definitely identified, but its fatty acid compositon was similar to that of phosphatidylcholine. However, it has an unusually high content of hexadecenoic acid, a fatty acid not common in the major erythrocyte phospholipids. 9. Accumulation of 1,2-diacyglycerol also occurred in energy-starved cells, even in the absence of calcium; in this case it appeared to be produced by phosphatidate breakdown.

Leaf Growth in Relation to ATP Levels in Water Stressed Corn Plants1

The cell water potential, turgor potential, leaf elongation rate, adenylate energy charge, and concentrations of ATP and free amino acids of elongating corn leaves were monitored during a 48 hour period after the induction of water stress to investigate early physical and metabolic changes. A decrease in cell turgor and rate of leaf elongation induced by the gradually decreasing leaf water potential from −2.6 to −18.0 bars over the 48 hour period under continuous light resulted in a 40% decrease in the ATP concentration of the elongating zone within 3 hours, and a 25 percent decrease in the adenylate energy charge within 12 hours. The concentration of free amino acids increased about 20% during the first 6 hours and over 250% during the 48 hours. The possible significance of the decrease in ATP concentration of the elongating leaf induced by water stress during a diurnal stress cycle is discussed.

Induction of ovalbumin and conalbumin synthesis in immature chick oviducts by ethionine.

PROLONGED feeding of rats with ethionine results in a high frequency of hepatic carcinoma. Ethionine induces other pathological manifestations in other organs and tissues of experimental animals1, and some of its effects can be reversed rapidly by the administration of methionine. At the molecular level ethionine induces a rapid decrease in hepatic ATP concentration2–4, followed by inhibition of RNA5 and protein synthesis6. The decrease in ATP concentration is due to formation of S-adenosyl-ethionine7, an inhibitor of tRNA methyltransferases8,9, and so tRNA isolated from the liver of rats injected with adenine and ethionine is hypomethylated10,11. It is well established that several eukaryotic mRNAs are methylated on the 5′ terminus and that such modification is essential for translation12,13. To study the mechanism of methylation we attempted to produce a specific methyl-deficient mRNA by the administration of ethionine. To monitor the effectiveness of the deprivation of methyl groups the synthesis of hormone-induced ovalbumin in immature chick oviducts was followed. Administration of oestrogen to immature chicks causes cytodifferentiation and growth of the primitive oviduct14–17. The tubular gland cells synthesise ovalbumin, conalbumin, ovomucoid and lysozyme which comprise 85–90% of the egg-white proteins18. The continuous presence of oestrogen is required for sustained synthesis of these proteins in immature chicks; withdrawal of oestrogen is accompanied by a gradual decline in cell-specific protein synthesis as well as in the weight of the oviduct and the RNA content of the tissue17. But, the tubular gland cells in the oviduct magnum during withdrawal are retained although they do not synthesise cell-specific secretory proteins17,19. Readministration of hormone to chicks after withdrawal (secondary stimulation) results in restoration of cell-specific protein synthesis without concomitant need for DNA synthesis17. During these studies ethionine unexpectedly simulated the effects of the injection of oestrogen for secondary stimulation.

Restriction of receptor mobility and the agglutination of cells by concanavalin a

Summary Transformed hamster fibroblasts with a high ATP content which are not agglutinated by concanavalin A (ConA) can be induced to agglutinate by decreasing their ATP content, mixing of glutaraldehyde prefixed ConA-coated cells with unfixed uncoated cells, or mixing of unfixed ConA-saturated cells with uncoated cells. The decrease in ATP content required for a gain of ConA agglutination between unfixed cells, was associated with a decrease in the short range rapid lateral mobility (RLM) of ConA surface receptors and with an increased exposure of the surface membrane phospholipids. The transformed fibroblasts with a high ATP content were agglutinated by the lectin from wheat germ (WGA) and after neuraminidase treatment also by the lectin from soybean (SBA). Agglutination by these two lectins was not effected by changes in the cellular ATP content. The half maximum rate ( V m ) of ConA-induced agglutination of cells with a low ATP content required 75% saturation of the cell receptors for ConA and was temperature sensitive. The half V m of agglutination between prefixed ConA-coated and unfixed uncoated cells, like the WGA- and SBA-induced agglutination of unfixed cells, required only about 15% saturation and was less temperature sensitive. The results indicate, that in cells with a high RLM of ConA receptors, agglutination induced by ConA required an appropriate degree of restriction of receptor mobility to allow the alignment of surface receptors that is required for agglutination. This restriction can be obtained by lowering the ATP content, mixing of prefixed lectin coated cells with unfixed uncoated cells, or saturating unfixed cells with ConA before mixing with unfixed uncoated cells. The results also suggest that the receptors for ConA are differently embedded in the lipid phase of the surface membrane than the receptors for WGA and SBA.

F factor-mediated inhibition of bacteriophage T7 growth: increased membrane permeability and decreased ATP levels following T7 infection of male Escherichia coli.

Cultures of uninfected and bacteriophage T7-infected male and female Escherichia coli were assayed for passive uptake of o-nitrophenyl-β-galactopyranoside and for ATP content. Relative to T7-infected female E. coli, infected male cells exhibit an increased permeability to o-nitrophenyl-β-galactopyranoside and a decrease in ATP content. Both phenomena are first observed between eight and 12 minutes after infection at 30°C and require (1) the presence of the F-factor genes which confer inhibition of phage growth and (2) phage-specific protein synthesis. These findings suggest that the abortive infection of male E. coli may represent a T7-induced structural alteration of the host membrane or a phage-induced failure of membrane-associated functions.

Effects of viscosity and ATP concentration on the movement of reactivated sea-urchin sperm flagella.

Spermatozoa from the sea urchin, Lytechinus pictus, can be demembranated with solutions containing Triton X-100 and 5mM-CaCl2 and reactivated in ATP solutions containing low concentrations (10(-9)M) Of free Ca2+ ion to give symmetrical bending wave movements, even at very low ATP concentrations. At ATP concentrations of 0.01-0.02 mM the reactivated spermatozoa have beat frequencies near 1 Hz, nearly normal bend angles, and wave-lengths about 50% longer than normal. 2. The effects of increased viscosity, obtained by addition of methyl cellulose to the reactivation solutions, on bend angle and beat frequency decrease with decreasing ATP concentration, and become almost undetectable at 0.01 mM ATP. On the other hand, the effect of increased viscosity on wavelength shows relatively little change with ATP concentration, although it is noticeably reduced at 0.01 mM ATP. 3. These observations suggest that the beat frequency at low ATP concentrations is determined by an intrinsic rate-limiting process, in contrast to the viscocity-limited behaviour at high beat frequencies. Quantitative agreement with the experimental results is obtained with a model in which ATP concentration and viscosity each determine the rates of one step in a two-step reaction cycle which determines the beat frequency. 4. Two other models which can qualitatively explain the effects of ATP concentration and viscosity on flagellar beat frequency fail to show quantitative agreement with the experimental results. In one of these models, ATP concentration determines the maximum rate of shear between filaments. In the other, ATP concentration determines a time delay which is required to bring the active moment into phase with the elastic moments which would be expected to dominate the bending resistance at low beat frequencies.

Active site titration and aminoacyl adenylate binding stoichiometry of aminoacyl-tRNA synthetases.

A simple, rapid, and economical procedure is described for the determination of the number of catalytically competent active sites on aminoacyl-tRNA synthetases based on the stoichiometry of aminoacyl adenylate formation. On mixing tRNA synthetase, cognate amino acid, (gamma-32P)ATP, and inorganic pyrophosphatase under suitable conditions there is an initial rapid stoichiometric "burst" (rate constant k1) of depletion of ATP as enzyme bound aminoacyl adenylate is formed. There is then an initially linear decrease in ATP concentration as the complex hydrolyzes (with rate constant k2) releasing enzyme to form further adenylate. Provided k2 less than k1 the initial burst gives the stoichiometry of aminoacyl adenylate formation. Complexes which are too unstable to be isolated by the usual gel or nitrocellulose disk filtration procedure may be assayed in this way. This technique has been applied to five highly purified aminoacyl-tRNA synthetases. The tyrosyl-tRNA synthetase from Bacillus stearothermophilus is shown to bind only one aminoacyl adenylate per dimer.

Effects of sodium fluoride and other metabolic inhibitors on basal and TSH-stimulated cyclic AMP and thyroid metabolism

Incubation of pig thyroid slices with NaF (50 m M), dicumarol (0.3 m M), iodoacetate (0.5 m M), and dinitrophenol (1 m M) significantly decreased ATP concentrations but did not diminish the basal levels of cyclic AMP, indicating that ATP concentrations may not be crucial in maintaining cyclic AMP. In pig and dog thyroid slices, NaF (50 m M) actually increased cyclic AMP concentrations. The increase was much less than that induced by 10 mU/ml TSH and was not observed in slices of beef or human thyroid tissue. Despite this increase in cyclic AMP concentrations, NaF (50 m M) did not augment glucose oxidation or 32P incorporation into phospholipids in pig thyroid slices. These results indicate that the stimulatory actions of NaF on the thyroid are probably not mediated by cyclic AMP. NaF, dicumarol, iodoacetate, and dinitrophenol all inhibited the increased cyclic AMP induced by TSH, indicating that ATP concentrations may be important for the effects of TSH. The metabolic inhibitors also inhibited the stimulation of glucose oxidation and 32P incorporation into phospholipids induced by TSH. Such inhibition parallels their effect on TSH stimulation of cyclic AMP but could also reflect the dependence of stimulation of these metabolic parameters on ATP concentrations.

Effect of hemorrhagic shock on tissue adenine nucleotides in conscious rats.

Hemorrhagic shock was produced in conscious rats by cannulating the subclavian artery and bleeding the animals to a mean arterial pressure of 40 mm Hg which was maintained for 1 (early shock) or 2 h (late shock). Analysis of tissues showed that there was a significant decrease in ATP and ADP levels in liver and kidney in early and late shock. Associated with the decrease in ATP and ADP levels were increases in AMP and P1 levels. In contrast to the above organs, adenine nucleotides and creatine phosphate levels of skeletal muscle did not decrease in early shock but a significant reduction of these compounds was observed in late shock. The decrease in ATP content was greater in liver and kidney than in skeletal muscle. The present experiments indicate that there is a decrease in the energy available to tissues during severe hemorrhagic shock. This could be due to decreased biosynthesis, to continuing or increased utilization of the nucleotides, or to both.

Rapid Phytochrome-mediated Changes in Adenosine 5′-Triphosphate Content of Etiolated Bean Buds

This study was designed to determine the effects of red and far red irradiation on ATP metabolism in etiolated bean buds (Phaseolus vulgaris L. var. Red Kidney). Compared to dark controls, red irradiated buds show an initial decline in ATP content at 15 seconds following a 5-minute irradiation. ATP content then rapidly rises to a peak at 1 minute, and then slowly returns to the baseline. The 1-minute promotion of ATP content is red/far red reversible. Acetylcholine does not appear to mimic red light in this system; it causes a marked decrease in ATP content.

Metabolic effects of hypoglycemic sulfonylureas—III: Effect of chlorpropam1de on energy metabolism and ketogenesis in the isolated perfused rat liver

The effects of 1 and 5 mM chlorpropamide were studied in the isolated perfused liver from fasted rats in the absence of exogenous substrate. 1 mM chlorpropamide caused a slight, but not significant, decrease in ATP content and in ATP/ADP ratio in the liver; 5 mM chlorpropamide caused a significant decrease in ATP, total adenine nucleotides and ATP/ADP ratio and an increase in ADP, AMP and inorganic phosphate content. The redox states of both cytosol and mitochondria, as measured by the lactate/pyruvate and β-hydroxybutyrate/acetoacetate ratios, respectively, were unaffected. However, there was a significant and dose dependent decrease in acetoacetate and β-hydroxybutyrate content. Livers perfused with 1 mM chlorpropamide showed a slight, but not significant, increase in oxygen uptake; 5 mM chlorpropamide had a biphasic effect: an initial stimulation, followed by a progressive inhibition of oxygen uptake. Ketone body production measured in the perfusion medium was inhibited by 1 and 5 mM chlorpropamide. No influenee could be seen on ureogenesis. The measured rates of oxygen uptake by the liver compared with the changes in oxygen uptake calculated from the reduced ketogenesis indicate a mixed effect of chlorpropamide on the perfused liver; (1) a decreased oxidation of endogenous triglycerides and (2) an increased oxidation of acetyl coenzyme A through the tricarboxylic acid cycle. The present findings suggest that chlorpropamide acts on the perfused liver as an uncoupling agent and as an inhibitor of the hepatic triglyceride lipase. (Glycerol ester hydrolase, EC 3.1.1.3.) Both effects could be related.

Ionic control of renal gluconeogenesis III. The effects of changes in pH, pCO 2, and bicarbonate concentration

The effect of changing pH, pCO 2 and HCO 3 − concentration upon the formation of glucose from a variety of substrates, and the rates of decarboxylation of these substrates was studied in isolated rat renal tubules. A decrease in pH whether brought about by an increase in pCO 2 or a decrease in HCO 3 − concentration led to an increase in glucose production from citrate, glutamine, glutamate and α-ketoglutarate but not from malate or pyruvate. Conversely an increase in pH, whether due to an increase in HCO 3 − concentration or a decrease in pCO 2, led to an inhibition of glucose formation from all substrates. An increase in pH brought about by an increase in HCO 3 − concentration produced a greater inhibition than an increase in pH brought about by a decrease in pCO 2. In both instances alkalosis was associated with a decrease in cellular ATP content. In the case of both citrate and α-ketoglutarate, the rate of substrate disappearance followed the same pattern as glucose production, but in the case of malate and pyruvate their rates of disappearance decreased with a fall in pH even though glucose formation did not change, and their rates of disappearance did not change when pH was increased even though glucose production fell markedly. A change in total HCO 3 − concentration from 30 to 15 mM, maintaining a constant pH of 7.2±0.02, resulted in an increase in glucose formation from citrate, glutamine, and α-ketoglutarate but not from malate or pyruvate. This increase was less than that seen after the pH fell. When HCO 3 − concentration was raised from 30 to 60 mM, at constant pH, there was a fall in rate of glucose formation from all substrates. This fall was comparably to that seen in respiratory alkalosis but less than that seen in metabolic alkalosis. These data, plus data from measurements of metabolic concentrations under different conditions of pH, pCO 2 and HCO 3 − concentration and measurements of substrate decarboxylation in both tubules and isolated renal mitochondria, led to the conclusions that when pH is lowered either by a fall in HCO 3 − or an increase in pCO 2, the major regulator of gluconeogenesis is H + concentration which acts to inhibit citrate synthetase and activate both isocitrate and succinate dehydrogenase, all mitochondria enzymes. Conversely, a decrease in H + concentration could account for only part of the changes observed in alkalosis. The fall in H + concentration led to a decrease in ATP concentration which led in turn to an inhibition of phosphoglycerate kinase. In addition, an increase in HCO 3 − concentration has a specific inhibitory effect upon phosphoenolpyruvate carboxykinase, a cytosolic enzyme.

Actions of 2-methyl-1,4-naphthoquinone in Bacillus cereus

Bacillus cereus was used to study the biochemical basis of the antibacterial action of 2-methyl-1,4-naphthoquinone (menadione). At 2.5 μM, menadione doubled the generation time of logarithmic phase cultures of B. cereus. The effect of the drug on the incorporation of labeled precursors for macromolecular synthesis was compared at equivalent turbidities. Incorporation of [ 14C]adenine and [ 14C]uracil into RNA was immediately inhibited. In contrast, incorporation of [ 14C]adenine into DNA, [ 14C]- l-lysine into protein, and [ 14C]diaminopimelic acid into cell wall was depressed only in accordance with decreased cell mass. RNA content of inhibited cells was also decreased when measured by the colorimetric orcinol method. Sucrose density gradient analysis of cell-free extracts from control and drug-treated cells labeled with [ 14C]-uracil showed that the drug effect was not specific for any particular RNA-containing fraction in that the incorporation of [ 14C]uracil was decreased in all macromolecular components, including ribosomes, ribosomal subunits and soluble RNA. Menadione produced an immediate dose-dependent decrease in ATP content of inhibited cells. Although other possibilities cannot be excluded, the growth-inhibitory effect of menadione in B. cereus might be due to decreased ATP levels causing a “shift-down” response in which growth retardation is characterized by a selective inhibition of RNA compared to DNA, protein or cell wall synthesis. Menadione also decreased the acid-soluble sulfhydryl content of inhibited cells. However, the mechanisms of this depletion as well as the relationship of this effect to inhibition of RNA synthesis and growth is unclear. Reduced glutathione, but not glutathione disulfide prevented the growth-inhibitory effect of menadione. Thiodione, a naphthoquinone thioether formed by reaction of reduced glutathione and menadione, did not inhibit the growth of B. cereus.

Rapid Respiratory Changes Due to Red Light or Acetylcholine during the Early Events of Phytochrome-mediated Photomorphogenesis

Two millimeter long secondary root tips of etiolated mung bean (Phaseolus aureus) plants were given 4 minute consecutive treatments of darkness, red light, far red light, and acetylcholine during darkness. We studied the effects of these treatments on exogenous (H(+)) changes, ATP utilization, O(2) uptake, P(1) levels, and ATPase activity. Red light and acetylcholine increased the level of P(1), O(2) uptake, and exogenous H(+), but decreased ATP concentrations. Darkness and far red light caused the amount of ATP to increase and decreased the O(2) uptake and P(1) level. O(2) uptake of both excised root tips and isolated mitochondria was promoted by acetylcholine levels of the same order of magnitude that promoted the other photomimetic phenomena. ADP-O ratios indicated that acetylcholine did not cause an appreciable decrease in ATP synthesis. The total ATPase activity remained constant throughout all treatments. Ouabain caused no adhesion to negatively charged glass in the dark, while the inhibitors valinomycin, atractyloside, digitoxin, gramicidin, and oligomycin caused immediate adhesion. All of the inhibitors prevented release from the glass. In red light ouabain increased adhesion, whereas the other inhibitors caused caused immediate and complete adhesion.These data seem to imply that one of the functions of the phytochrome-mediated response to red light in roots, regulated by acetylcholine, is to cause the rapid utilization of ATP pools; far red light appears to inhibit this utilization.

Regulation of cellular respiration by thyroid hormone. Spectroscopic evidence of mitochondrial control in intact rat liver

In order to elucidate the role of the mitochondrial control (coupling) mechanism in the regulation of respiration in intact liver, we investigated perfused rat livers using direct spectrophotometry. The acute effects of fructose on hepatic concentrations of adenine nucleotides, inorganic phosphate, and citrate were also studied. In hypothyroid rat livers fructose caused a small stimulation of respiration, followed by marked inhibition lasting about 10 min. During the respiratory inhibition the redox state of the flavoproteins was reduced but that of cytochrome b was markedly oxidized. In euthyroid rats fructose first stimulated hepatic oxygen consumption, but the subsequent inhibition of respiration was much smaller than that in hypothyroid rats. During the inhibitory phase flavoproteins were reduced, but the oxidation of cytochrome b was significantly less than in hypothyroid rats. In hyperthyroid rat liver, fructose stimulated respiration without any subsequent inhibition. Flavoproteins and all cytochromes were reduced, and no transient oxidation was observed in contrast to hypo- and euthyroid rats. In all groups of rats fructose induced a prompt decrease in hepatic concentrations of ATP and inorganic phosphate. Fructose did not induce any acute changes in the concentrations of citrate, but in hyperthyroid rat liver the citrate concentration was three times greater than in hypo- or euthryoid liver. These results suggest that, in intact tissue also, mitochondrial control is important in the regulation of energy metabolism during the metabolism of fructose. Thyroxine treatment seems to loosen this coupling, perhaps by increasing the concentration and oxidation of fatty acids in the liver.