Phosphate Uptake in an Obligately Marine Fungus II. Role of Culture Conditions, Energy Sources, and Inhibitors

Phosphate uptake in the obligately marine fungus, Thraustochytrium roseum, is maximal at pH 7.5 to 7.8, is dependent on temperature, and varies with phosphate concentration. Pyruvate and succinate stimulate phosphate uptake, although they do not increase respiration. The uncoupling agents, 2,4-dinitrophenol and dicoumerol, inhibit phosphate uptake but stimulate oxygen consumption only in the presence of NaCl. Oligomycin inhibits both processes. Among the inhibitors of protein synthesis, chloramphenicol reduces phosphate uptake without affecting respiration. Puromycin is unique in that it greatly enhances phosphate uptake and abolished the lag period associated with this phenomenon. It does not affect respiration.     

Uncoupling of energy-linked functions of corn mitochondria by linoleic Acid and monomethyldecenylsuccinic Acid

Linoleic acid and monomethyldecenylsuccinic acid were tested as uncoupling agents for energy linked functions of corn mitochondria. 2,4-dinitrophenol was used as a standard for comparison. Both compounds uncoupled oxidative phosphorylation, released oligomycin-blocked respiration, and accelerated adenosine triphosphatase. Linoleic acid uncoupled calcium-activated phosphate accumulation and the increase in light scattering that accompanies the accumulation. Unlike dinitrophenol, linoleic acid at 0.1 mm had a destructive effect on membrane semipermeability. Kinetic studies indicated that dinitrophenol and linoleic acid compete with phosphate for active sites in oxidative phosphorylation.     

The Relation of Phosphate Metabolism to Oxidative Assimilation of Acetate by Euglena gracilis var. bacillaris. Effects of 2,4-Dinitrophenol*

SYNOPSIS. Oxidative assimilation of acetate by Euglena gracilis var. bacillaris occurs without appreciable net transfer of inorganic phosphate between cells and medium. Low concentrations of the “uncoupling agent” 2,4-dinitrophenol (2–5 μM, pH 5) stimulate acetate oxidation and inhibit acetate assimilation. These dinitrophenol concentrations have no measurable effect on edogenous respiration. Higher concentrations inhibit respiration on acetate, and still higher concentrations inhibit endogenous respiration. Dinitrophenol concentrations which stimulate acetate oxidation produce no measurable change in the total ATP content of the euglena cells. Higher concentrations (50–100 μM) cause progressive decrease in the ATP content. A hypothetical mechanism is proposed which accounts for the normal, constant oxidation-assimilation ratio for acetate as the result of coupling of oxidative ATP synthesis with assimilatory ATP consumption. The data are consistent with this mechanism if it is additionally assumed that oxidation and assimilation of acetate occur in an intracellular “compartment” separated from other sources and sinks of ATP.     

Leaf respiration: Possible cause of stimulation after low temperature treatment

Uncoupling of oxidative phosphorylation by 2,4 dinitrophenol stimulated leaf respiration rate measured as CO₂ production or O₂ uptake after low temperature treatment.     

Preparation and some properties of a suspension of fragmented tubules from rat kidney

A method is described whereby short fragments of rat kidney tubule were obtained when kidney slices were gently dispersed by exposure to collagenase and hyaluronidase. When suspended in buffered saline the fragmented tubules respired actively over a period of several hours, the rate of oxygen consumption being proportional to the amount of cell protein. Oxygen uptake was stimulated by the addition of glucose, lactate, butyrate, alpha-oxoglutarate and other substrates and was decreased by the omission of Ca(2+) from the suspending medium. With alpha-oxoglutarate as the added substrate, dinitrophenol strongly stimulated oxygen uptake. Dinitrophenol had a less-marked stimulatory effect when glucose was the added substrate, and inhibited respiration in the absence of added substrate. Oligomycin inhibited respiration and this inhibition was partially reversed by dinitrophenol. Fragmented tubules synthesized glucose from lactate at a high rate but this capacity for gluconeogenesis was abolished by dinitrophenol and by physically damaging the cells.     

Reserpine as an uncoupling agent

1. Reserpine, like the uncoupling agent, 2,4-dinitrophenol prevents oxidative phosphorylation but stimulates the rate at which oxygen is reduced.

2. Both reserpine and 2,4-dinitrophenol fail to stimulate oxygen uptake by isolated mitochondria in the presence of arginine.

3. Both 2,4-dinitrophenol and reserpine induce proton permeability in the mitochondrial membrane so that H⁺ is absorbed from the suspending medium.

4. When the reaction system contains reserpine, accumulation of Ca²⁺ by mitochondria is inhibited.

5. Reserpine decreases both ADP:O and P:O ratios which strongly suggest that reserpine is an uncoupling agent.     

Uncoupling of respiration-linked contraction in corn mitochondria

Respiration-linked contraction of corn mitochondria is not noticeably reduced by low, uncoupling concentrations of dinitrophenol. However, if a contraction/respiration ratio is calculated, the contraction proves to be uncoupled. Previous statements that contraction cannot be uncoupled from respiration are in error.

The uncoupling of contraction is consistent with the concept that dinitrophenol attacks a primary non-phosphorylated high energy intermediate (I~X). It is proposed that this intermediate is linked to some contractile mechanism such that the degree of contraction reflects the level of intermediate.

     

Sulfate Uptake and Respiration of Aging Potato Discs Modified by Malonic Acid and Ultraviolet Radiation

The time course of sulfate uptake in relation to respiration is shown for aging slices of potato, Solanum tuberosum L. cv. Russet. For comparative purposes, a time course for phosphate uptake is also shown. Malonic acid depressed both the respiratory rise and the sulfate accumulating ability of aging discs. A low dose of ultraviolet radiation given at the onset of aging reduced sulfate uptake by one-half without lowering respiration. We hypothesize that sulfate uptake is controlled by at least two mechanisms: one associated with oxidative metabolism and the other mediated by protein synthesis.     

Effects of the Commercial Polychlorinated Biphenyl Mixture Aroclor 1242 on Growth, Viability, Phosphate Uptake, Respiration and Oxygen Evolution in Scenedesmus

The commercial polychlorinated biphenyl (PCB) mixture Aroclor 1242 was tested regarding its effects on the uni-cellular green alga Scenedesmus obtusiasculus Chod. The algae were grown in liquid medium at PCB concentrations between 10 and 1000 ppb. Growth was inhibited at 300 ppb and above, viability was only affected at the highest concentration, 1000 ppb. Reduction of phosphate uptake was seen at 300 ppb and above. The inhibition of phosphate uptake had almost exactly the same appearance in light as in darkness. It is suggested that this inhibition is mainly caused by PCB action on the plasmalemma. At 800 ppb some experiments indicated an uncoupling of the oxidative phosphorylation. 1000 ppb inhibited both respiration and O₂ evolution.     

Respiratory Response of Acer pseudoplatanus Cells to Pyruvate and 2,4-Dinitrophenol

The endogenous respiration rate of unstarved cultured cells of Acer pseudo-platanus L. is markedly stimulated by 2,4-dinitrophenol. Pyruvate is also stimulatory but to a lesser degree than dinitrophenol. Exogenously supplied sugars cause no short-term stimulation. Pyruvate does not enhance the elevated rate of O₂ uptake in the presence of dinitrophenol but does cause additional CO₂ evolution. The endogenous concentration of pyruvate is elevated in the presence of dinitrophenol. These observations suggest that the rate of O₂ uptake by the unstarved intact cells is limited by the rate of glycolysis and that rate of glycolysis is regulated by the intracellular concentration of adenine nucleotides or inorganic phosphate. Dinitrophenol stimulation of endogenous respiration is due in part to an indirect acceleration of glycolysis but also to a more direct facilitation of oxidation in the presence of excess mitochondrial substrate.     

State of liver mitochondrial respiratory chain in rats with experimental toxic hepatitis

Polarographical determination of oxygen concentration has shown that in rats with experimental hepatitis induced by combined ethanol and CCl₄ administration for 4 weeks, the functioning of the hepatocyte mitochondrial respiratory chain is impaired. Development of liver pathology was accompanied by adipose dystrophy, fibrosis, and an increase of triglycerides and lipid peroxidation products in the liver tissue. The endogenous respiration rate in hepatocytes isolated from the pathologically altered liver was 34% higher than in the control. Cell respiration was not stimulated by the addition of the substrates malate and pyruvate with digitonine. An uncoupler of oxidation and phosphorylation, 2,4-dinitrophenol, increased the hepatocyte oxygen consumption rate by 37%, while addition of the inhibitor of the I complex, rotenone, decreased cell respiration in pathologically altered hepatocytes by 27%. The states 3 (V₃) and 4 (V₄) of mitochondrial respiration with malate + glutamate as substrates were found to be higher by 70% and 56%, respectively, as compared with the control level. When using malate + glutamate or succinate as substrates, V₃ and Vd (dinitrophenol respiration) in the toxic hepatitis hepatocyte mitochondria did not differ from the control, which indicates no uncoupling occurred of the oxidation and phosphorylation processes. Cytochrome c oxidase activity was elevated (+80%) as compared with the control. Administration of the hypolipidemic agent symvastatin simultaneously with ethanol and CCl₄ resulted in a reduction of the degree of liver adipose dystrophy, prevented activation of lipid peroxidation, and decreased the hepatocyte endogenous respiration rate. Addition of malate + pyruvate, dinitrophenol or rotenone produced oxygen consumption changes similar to those in the control. However, in mitochondria isolated from the pathologically altered liver, symvastatin induced an uncoupling effect on the respiratory chain in the presence of the substrates malate + glutamate, but did not change the cytochrome c oxidase activity. We suggest that functioning of the NCCR complex in the hepatocyte mitochondria of animals with experimental toxic hepatitis is impaired, which leads to an intensive superoxide anion production at the level of this complex. Under these conditions, the defect of the NADH-coenzyme Q-oxidoreductase is compensated by functioning of other complexes of the respiratory chain (SCCR, coenzyme Q-cytochrome c-reductase, cytochrome c oxidase, and ATP-synthase activities).     

The ATP/ADP-antiporter is involved in the uncoupling effect of fatty acids on mitochondria

The ATP/ADP-antiporter inhibitors and the substrate ADP suppress the uncoupling effect induced by low (10-20 microM) concentrations of palmitate in mitochondria from skeletal muscle and liver. The inhibitors and ADP are found to (a) inhibit the palmitate-stimulated respiration in the controlled state and (b) increase the membrane potential lowered by palmitate. The degree of efficiency decreases in the order: carboxyatractylate (CAtr) greater than ADP greater than bongkrekic acid, atractylate. GDP is ineffective, Mg.ADP is of much smaller effect, whereas ATP is effective at much higher concentration than is ADP. Inhibitor concentrations, which maximally suppress the palmitate-stimulated respiration, correspond to those needed for arresting the state 3 respiration. The extent of the CAtr-sensitive stimulation of respiration by palmitate has been found to decrease with an increase in palmitate concentration. Stimulation of the controlled respiration by p-trifluoromethoxycarbonylcyanide phenylhydrozone (FCCP) and gramicidin D at any concentrations of these uncouplers is CAtr-insensitive, whereas that caused by a low concentrations of 2,4-dinitrophenol and dodecyl sulfate is inhibited by CAtr. The above effect of palmitate develops immediately after addition of the fatty acid. It is resistant to EGTA as well as to inhibitors of phospholipase (nupercain) and of lipid peroxidation (ionol). Moreover, palmitate accelerates spontaneous release of the respiratory control, developing in rat liver mitochondria under certain conditions. This effect takes several minutes, being sensitive to EGTA, nupercain and ionol. Like the fast uncoupling, this slow effect is inhibited by ADP but CAtr and atractylate are stimulatory rather than inhibitory. In artificial planar phospholipid membrane, palmitate does not increase the membrane conductance, FCCP increases it strongly and dinitrophenol only slightly. In cytochrome oxidase proteoliposomes, FCCP, gramicidin and dinitrophenol (less effectively) lower, whereas palmitate enhances the cytochrome-oxidase-generated membrane potential. In this system, monensin substitutes for palmitate. It is concluded that the ATP/ADP antiporter is somehow involved in the uncoupling effect caused by low concentrations of palmitate and, partially, of dinitrophenol, whereas uncoupling produced by FCCP and gramicidin is due to their action on the phospholipid part of the mitochondrial membrane. A possible mechanism of this effect is discussed.     

Hepatocyte mitochondrion electron-transport chain alterations in CCl<Subscript>4</Subscript> and alcohol induced hepatitis in rats and their correction with simvastatin

The goal of this study was to examine the state of hepatocyte mitochondrial respiratory chain of rats with toxic hepatitis induced by CCl₄ and ethanol. Oxygen consumption by hepatocytes and mitochondria was determined. Endogenous oxygen consumption by pathological hepatocytes was 1.3-fold higher compared with control. Rotenone resulted in 27% suppression of respiration by pathological hepatocytes whereas 2,4-dinitrophenol produced a 1.4-fold increase of respiration. States 3 and 4 of mitochondrial respiration with malate and glutamate were found to be higher as compared to control. State dinitrophenol and state 3 respirations were similar within every group of animals when being tested with malate and glutamate or succinate. Cytochrome c oxidase activity in hepatitis was 1.8-fold higher compared with control. Simvastatin administration resulted in a decrease in hepatocyte endogenous respiration in hepatitis. The presented data lead to the assumption that the increased oxygen consumption by the respiratory chain of pathological mitochondria to be linked mainly with the altered function of complex I.     

Photooxidative damage to the photosynthetic apparatus during chilling

Short periods of chilling in the presence of light (up to 6 h: 1°C; 270 W/m²) decreased the subsequent apparent photosynthesis and chlorophyll fluorescence of leaf discs of Cucumis sativus L. cv. Kleine Groene Scherpe. The extent of the injury depended on the duration of the chilling pretreatment. After 6 h the subsequent apparent photosynthesis even reached a negative value, and it increased only slightly during the next 2 1/2 h. The decrease of apparent photosynthesis was not a consequence of increased dark respiration but was of photooxidative origin since the presence of both light and oxygen was required. Preincubation in the light for 2 h at 20°C sensitized leaf discs to subsequent photooxidation during chilling. Prompt and delayed chlorophyll fluorescence decreased simultaneously after chilling and light treatment. The corresponding decrease of photosynthesis and chlorophyll fluorescence is discussed in relation to primary photooxidative damage to the photosystems in the chloroplast membrane.     

Oxygen uptake and lactate production by Schistosoma mansoni cercaria, cercarial body and tail, and schistosomule.

1. Oxygen consumption by Schistosoma mansoni cercarial bodies varies, with the batch of organisms, the incubation media and the temperature (27-37 degrees C), from 27.4 +/- 3.4 to 55.0 +/- 4.8 microliters O2/mg larval protein per hr. It is proportional to the concentration of organisms incubated, up to 25,000/ml, as calculated from whole protein. 2. Oxygen uptake by cercariae is inhibited by 5.6 mM glucose in the incubation media, a concentration that stimulates the respiration of cercarial bodies. 3. No significant differences in the oxygen uptake were presented by cercarial bodies with and without glycocalyx or glandular secretions, or devoid of all of them. 4. Inhibitors of the Krebs cycle and the respiratory chain, and uncoupling agents influence the oxygen uptake by cercariae, cercarial bodies and schistosomules to the same extent. 5. The permeability change presented by transformed larvae had no influence on the excretion of lactate by cercarial bodies, which is about 0.3 mumoles/mg protein per hr and remains constant for 5 hr; under nitrogen, this amount increased 70%. Cercariae in anaerobiosis, however, excreted as much as 15 times more lactate than under air. 6. Lactic dehydrogenases of cercariae, cercarial bodies and tails, and schistosomules are of the muscle type and do not change during the transformation.     

Higher respiratory activity decreases mitochondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae

Increased replicative longevity in Saccharomyces cerevisiae because of calorie restriction has been linked to enhanced mitochondrial respiratory activity. Here we have further investigated how mitochondrial respiration affects yeast life span. We found that calorie restriction by growth in low glucose increased respiration but decreased mitochondrial reactive oxygen species production relative to oxygen consumption. Calorie restriction also enhanced chronological life span. The beneficial effects of calorie restriction on mitochondrial respiration, reactive oxygen species release, and replicative and chronological life span could be mimicked by uncoupling agents such as dinitrophenol. Conversely, chronological life span decreased in cells treated with antimycin (which strongly increases mitochondrial reactive oxygen species generation) or in yeast mutants null for mitochondrial superoxide dismutase (which removes superoxide radicals) and for RTG2 (which participates in retrograde feedback signaling between mitochondria and the nucleus). These results suggest that yeast aging is linked to changes in mitochondrial metabolism and oxidative stress and that mild mitochondrial uncoupling can increase both chronological and replicative life span.     

The mitochondrial uncoupler 2,4-dinitrophenol attenuates tissue damage and improves mitochondrial homeostasis following transient focal cerebral ischemia

Ischemic stroke is caused by acute neuronal degeneration provoked by interruption of cerebral blood flow. Although the mechanisms contributing to ischemic neuronal degeneration are myriad, mitochondrial dysfunction is now recognized as a pivotal event that can lead to either necrotic or apoptotic neuronal death. Lack of suitable 'upstream' targets to prevent loss of mitochondrial homeostasis has, so far, restricted the development of mechanistically based interventions to promote neuronal survival. Here, we show that the uncoupling agent 2,4 dinitrophenol (DNP) reduces infarct volume approximately 40% in a model of focal ischemia-reperfusion injury in the rat brain. The mechanism of protection involves an early decrease in mitochondrial reactive oxygen species formation and calcium uptake leading to improved mitochondrial function and a reduction in the release of cytochrome c into the cytoplasm. The observed effects of DNP were not associated with enhanced cerebral perfusion. These findings indicate that compounds with uncoupling properties may confer neuroprotection through a mechanism involving stabilization of mitochondrial function.     

Metabolic regulation in the senescing tobacco leaf: I. Changes in pattern of p incorporation into leaf disc metabolites

Changes in phosphate metabolism were explored in discs from tobacco (Nicotiana tabacum) leaves of three contrasting types: green leaves which were fully expanded and attached to the plant, leaves which had yellowed following excision and dark starvation, and leaves which had yellowed while attached to the plant. 2,4-Dinitrophenol at 10⁻⁵m stimulated the respiration rate of discs from green and yellow-detached leaves only slightly, but markedly stimulated that of discs from yellow-attached leaves. Following a 10-minute uptake period the incorporation of ³²P-orthophosphate into phosphate esters and lipids of discs from yellow-detached leaves was resistant to 2,4-dinitrophenol, whereas in discs from green and yellow-attached leaves it was inhibited by 2,4-dinitrophenol. Incorporation into a salt-soluble fraction containing unidentified nucleotide material showed converse behavior in that it was stimulated by 2,4-dinitrophenol in discs from green and yellow-attached leaves; in discs from yellow-detached leaves it was resistant to 2,4-dinitrophenol. In discs from yellow-detached and yellow-attached leaves there was a shift in the labeling pattern of phosphate esters toward increased label in hexose phosphates at the expense of adenine nucleotides, 3-phosphoglycerate, and phosphoenolpyruvate. It is concluded that incorporation into phosphate esters in discs from yellow-detached leaves is by substrate level phosphorylation coupled to enhanced aerobic glycolysis. In discs from yellow-attached leaves, on the other hand, incorporation depends on oxidation phosphorylation, and it is suggested that the shift in labeling pattern is caused by senescence-induced changes in activity of glycolytic enzymes.     

Phosphorylation in avocado fruit slices in relation to the respiratory climacteric

The rate of uptake of inorganic phosphate by tissue discs from both preclimacteric and climacteric peak avocados is linear for at least 60 minutes. The loss of ³²P upon excessive washing was much greater from peak than from preclimacteric tissue. Short incubation periods and, most important, rapid washing procedures are essential for meaningful comparisons.

Phosphate esterification proceeded at a much greater rate in climacteric than in preclimacteric tissue. The phosphorylation was sensitive to 2,4-dinitrophenol (DNP). The ADP to ATP ratio decreased materially with the advance of ripening. It was concluded that neither uncoupling nor acceptor control can account for the onset of the respiratory rise. Changes in permeability appear to play an important role in fruit metabolism during the climacteric.

     

Inhibition of nitrate and nitrite reduction by 2,4-dinitrophenol in ankistrodesmus

Summary As Kessler (1955, 1959) has shown, nitrite reduction by the green alga, Ankistrodesmus braunii is completely inhibited by 10^-3 m 2,4-dinitrophenol. However, although nitrite accumulates in the medium when cultures are supplied with nitrate and dinitrophenol, the reduction of nitrate is not completely insensitive to the inhibitor.Direct measurements show that 2,4-dinitrophenol inhibits nitrate disappearance from the medium by 65–80%. The degree of inhibition increases when the initial nitrate concentration is decreased.It is suggested that inhibition of nitrate assimilation by dinitrophenol is due to inhibition of an active uptake of nitrate by the cells, and that at high nitrate concentrations, a dinitrophenol-insensitive uptake process increases in importance.