Pyridine Nucleotide Levels Research Articles

Glucose Metabolism and Pyridine Nucleotide Levels in Potato Tubers Treated with Ethylene

Summary Ethylene's ability to enhance potato tuber respiration is well established. In this study the respiratory burst induced by ethylene was associated with changes in the metabolism of glucose and altered pyridine nucleotide levels. The data suggest that the pentose phosphate pathway was proportionally stimulated to a greater degree than the Embden-Meyerhoff-Parnas Tricarboxylic Acid pathway. The oxidized forms of the pyridine nucleotides were increased relative to the reduced forms, suggesting that these changes may playa role in directing glucose catabolism.

Perturbation in liver mitochondrial Ca 2+ homeostasis in experimental iron overload: a possible factor in cell injury

The functional state of isolated mitochondria and specifically the integrity of the inner membrane, were investigated in the liver of rats made siderotic by dietary supplementation with carbonyl iron. The concentration of iron in the hepatic tissue increased progressively up to nearly 40 days and reached a steady-state level. When the iron content reached a threshold value (higher than 90 nmol/mg protein) the occurrence of in vivo lipid peroxidation in the mitochondrial membrane was detected. This process did not result in gross alterations in the mitochondrial membrane, as indicated by electron microscopy, phosphorylative capability and membrane potential measurements. On the contrary, the induction of lipoperoxidative reaction appeared to be associated with the activation of Ca2+ release from mitochondria. This was shown to occur as a consequence of rather subtle modifications in the inner membrane structure via a specific efflux route, which appeared to be linked to the oxidation level of mitochondrial pyridine nucleotides. The induction of this Ca2+ release from iron-treated mitochondria resulted in enhancement of Ca2+ cycling, a process which dissipates energy to reaccumulate into mitochondria the released Ca2+. The perturbation in mitochondrial Ca2+ homeostasis reported here may be a factor in the onset of cell damage in this experimental model of hepatic iron overload.

Effect of riboflavin deficiency on sorbitol pathway in rat lens

The sorbitol pathway comprising of NADPH dependent aldose reductase (AR) and NAD + dependent sorbitol dehydrogenase (SDH) which convert glucose to sorbitol and then to fructose, respectively was studied in riboflavin deficient rat lens. The activities of AR and SDH were increased significantly in lenses from riboflavin deficient rats as compared to both ad libitum and pair-fed control groups of rats. While the ratio of NADPH to NADP +, and the levels of NADPH were also increased significantly in the riboflavin deficient rat lenses over the riboflavin supplemented (both ad libitum and pair-fed) rat lenses, the levels of total pyridine nucleotides (NADPH+NADP +) in deficient rat lenses were significantly higher than those from ad libitum controls alone. There were, however, no significant differences in the levels of sorbitol among the three groups of rats. The data suggest that the sorbitol pathway is enhanced in rat lens during riboflavin deficiency.

Adenine and pyridine nucleotide levels during calcium-induced conidiation in Penicillium notatum.

Concentrations of adenine and pyridine nucleotides and the associated charge values were examined in extracts of mycelium of Penicillium notatum during vegetative growth and reproductive development promoted by the addition of Ca2+ (10 mmol dm-3). The significant increase in adenylate energy charge promoted by Ca2+ was due to a fall in intracellular AMP and a concomitant rise in ATP concentration. Intracellular concentrations of NADH and NAD fell within 1 h of the addition of Ca2+. The catabolic reduction charge was unchanged by Ca2+ whilst the anabolic reduction charge increased in Ca2+-induced mycelium due to lowered intracellular NADP concentration. Reduced concentration of NADPH in Ca2+-induced mycelium, relative to the vegetative controls, lowered the phosphorylated nucleotide fraction. The results are discussed in relation to metabolic economy during morphogenesis in P. notatum.

Cellular energy levels and their effect on male cell abortion in cytoplasmically male sterile lines of Petunia hybrida

Using an HPLC method it has proved possible to follow the levels of adenine and pyridine nucleotides in the anthers of normal and cytoplasmically male sterile (CMS) lines of Petunia hybrida. Well before the appearance of any structural differences, anthers of CMS plants begin to show lower ATP/ADP ratios. In anthers, as in other non-photosynthetic plant tissues, there is a strong correlation between the ATP/ADP ratio and levels of NADPH and, when NADPH was assayed in fertile and CMS anthers, very dramatic differences were discovered. In male fertile plants, the NADPH/NADP ratio differs strikingly between somatic and reproductive tissues and reaches a peak in anthers at the early prophase of meiosis. The ratio in male sterile anthers remains largely unchanged from that normally seen in somatic tissues over this period. A cytochemical localisation technique revealed that, at the stage of development in question, the major reserves of anther NADPH are held within the tapetal cells and that levels within CMS tapeta are strikingly lower than those of normal plants. These findings are discussed in the perspective of theories proposed to explain the operation of CMS, and also in terms of the now conclusive evidence that the genetical basis of CMS lies in changes in the organisation of the mitochondrial genome.

Determination of intracellular pyridine nucleotide levels by bioluminescence using anaerobic bacteria as a model

An assay for the determination of NAD(P) + and NAD(P)H in extracts from the obligate anaerobe bacterium Thermoanaerobacter finnii is developed and the strategy for this development is described. This assay performed with constant FMN reductase (EC 1.6.8.1) and luciferase (EC 1.14.14.3) concentrations has been shown to detect as low as 1 pmol pyridine nucleotide. With this assay recovery, efficiency of extraction, reliability, and detection limit of the procedures were determined. To our knowledge this is the first bioluminometric assay for the determination of pyridine nucleotide levels in bacterial extracts.

Light inhibition of mitochondrial respiration in a mutant of Chlamydomonas reinhardtii devoid of ribulose-1,5-bisphosphate carboxylase/oxygenase activity

The effect of light on mitochondrial respiration has been investigated in Chlamydomonas reinhardtii rcl-u-1-10-6C, a mutant devoid of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity. No CO 2 uptake was observed in the light, confirming that there was no Rubisco activity, but the CO 2 evolution rate was diminished by 65 to 80%. This inhibition was ascribable to a decrease in the tricarboxylic acid cycle (Krebs cycle) activity. At the same time, O 2 evolution associated with stimulation of the O 2 uptake appears. Darkness or addition of DCMU fully reversed the effect of light, indicating that the inhibitory process is linked to photosystem activities. Levels of pyridine nucleotides (NAD(H) and NADP(H)) and adenine nucleotides (ATP and ADP), the most probable mediators of the interaction between photosynthesis and respiration, were measured in dark and in light. During a dark to light transition the level of NADPH increased significantly whereas the NAD(H) pool remained almost fully oxidized. The level of ADP was always extremely low. These results suggest that the inhibition of Krebs cycle activity is due to a competition for cytosolic ADP between chloroplastic photophosphorylations and oxidative phosphorylations.

Iron-induced Ca 2+ release from rat liver mitochondria via H 2O 2 metabolism

The addition of iron(III)-gluconate complex to isolated rat liver mitochondria induced a net efflux of Ca 2+ not inhibitable by ruthenium red, which resulted in enhancement of Ca 2+ cycling and a simultaneous membrane potential drop. Iron caused extensive oxidation of mitochondrial pyridine nucleotides. However, it failed to induce appreciable changes in either the oxidation level of pyridine nucleotides or the membrane potential in mitochondria isolated from rats fed with a selenium-deficient diet. Iron also induced a transient increase in the mitochondria cyanide-insensitive oxygen consumption. This effect was largely prevented by the addition of the hydrogen peroxide scavenger catalase. It was concluded that iron induced the activation of a specific Ca 2+ efflux pathway via the oxidation of pyridine nucleotides due to the hydrogen peroxide metabolism by the glutathione enzyme system.

Changes in pyridine nucleotide levels alter oxygen consumption and extra-mitochondrial phosphates in isolated mitochondria: A 31P-NMR and NAD(P)H fluorescence study

Isolated rat-liver mitochondria were used to study the relation between mitochondrial NADH levels, oxygen consumption (QO 2), and extra-mitochondrial phosphates. Alterations in NADH and QO 2 were accomplished by incubating mitochondria with different substrates or varying amounts of exogenous ATPase while monitoring QO 2 and NAD(P)H fluorescence. Two sets of conditions were studied: (1) in the presence of excess ADP and inorganic phosphate, an increase in NAD(P)H fluorescence was associated with a linear increase in QO 2; (2) when QO 2 was driven by the steady-state hydrolysis of ATP by exogenous ATPase, increases in QO 2 were associated with proportional decreases in NAD(P)H fluorescence. For all substrates tested this relation was linear; however, the slope was substrate dependent. Different substrates were able to maintain different NAD(P)H levels at the same QO 2. To investigate this further, effects of changing substrates at constant QO 2 on NAD(P)H and extra-mitochondrial phosphates were determined. Addition of glutamate + malate to mitochondria respiring on citrate caused a 50% increase in NAD(P)H fluorescence, a 41% decrease in ADP, and a 30% decrease in inorganic phosphate. Similar changes for the substrate jump, pyruvate + malate to glutamate + malate were found. Finally, it was determined that a linear relation holds between increases in NAD(P)H fluorescence and increases in QO 2 when substrates were varied at constant, physiologic levels of extra-mitochondrial ADP. These results indicate that QO 2 depends on NAD(P)H levels as well as on extra-mitochondrial phosphates over a wide range of respiratory rates.

Alkaline extraction and reverse-phase high-performance liquid chromatography of adenine and pyridine nucleotides in human platelets

The levels of adenine (ATP, ADP, AMP) and pyridine (NAD, NADH) nucleotides in human platelets have been measured by a simple and reproducible method. A rapid alkaline extraction allows a complete recovery of the compounds concerned. The metabolic ATP and ADP in the cytosolic fraction, the amount released upon thrombin stimulation, and the ADP bound to F-actin have also been evaluated. Analysis was performed by reverse-phase, isocratic high-performance liquid chromatography on a 5-μm Lichrosorb RP-18 column with uv detection at 254 nm.

The metabolite ratio as a function of chloral hydrate dose and intracellular redox state in the perfused rat liver.

Chloral hydrate (CH), an intermediate metabolite of trichloroethylene, is reduced to trichloroethanol (TCE) by alcohol dehydrogenase and aldehyde reductase, and is also oxidized to trichloroacetic acid (TCA) by the nicotinamide adenine dinucleotide (NAD)-dependent enzyme, CH dehydrogenase. Alcohol dehydrogenase requires reduced NAD (NADH), aldehyde reductase requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) and CH dehydrogenase requires NAD to complete the reaction. It is unclear which reaction is predominant at the physiological redox level in intact liver cells. To study this question, we perfused the livers of well-fed rats with Krebs-Ringer buffer solution containing 0.1 mM pyruvate/1.0 mM lactate. The levels of TCE and TCA in the effluent were measured by gas chromatography, and the fluorescence of reduced pyridine nucleotides was measured with a surface fluorometer. When a low concentration (below 0.25 mM) of CH was administered, more TCA than TCE was produced. When a high concentration of CH was administered (over 0.5 mM), TCE production was greater. Reduced pyridine nucleotides decreased inversely with the CH concentration. Even at low CH concentrations, pyridine nucleotides were not reduced. When 10 mM lactate was added to the perfusate in order to reduce the pyridine nucleotides in the liver cells, the TCE/TCA ratio increased. On the other hand, the TCE/TCA ratio tended to fall following the addition of 5.0 mM pyruvate. In conclusion, the TCE/TCA ratio was altered according to the concentration of CH, and to the redox level of pyridine nucleotides in the liver.

Changes in the level of oxidized and reduced pyridine nucleotides during cold acclimation of winter rape plants

Prolonged cold (2°C) treatment of winter rape plants (Brassica napus L. var. oleifera L. cv. Górczański) markedly modified the pattern of leaf growth and brought about changes in the level of pyridine nucleotides already during the first few days of treatment. The NAD+, NADP+ and NADPH levels markedly increased but there was practically no effect on the NADH level. Changes in the respective nucleotide levels were reflected by changes in anabolic and catabolic reduction charges. The former increased by 70%, whereas the latter decreased by 44%. Alterations in pyridine nucleotide levels and the reduction charges are discussed in terms of possible mechanisms involved, as well as in terms of their role in plant adaptation to cold.

Interaction between leukotriene D4 and adenosine or iloprost in the isolated working guinea‐pig heart: prevention of the leukotriene D4 effect

The interaction between leukotriene D4 and adenosine or the prostacyclin analogue iloprost was studied in isolated guinea-pig hearts. Adenosine (1 X 10(-6) M) or iloprost (5 X 10(-8) M) abolished or greatly attenuated the vasoconstrictive effect of leukotriene D4 over a wide dose range of leukotriene D4 (0.01-1000 ng), and myocardial ischemia as a consequence of coronary insufficiency completely disappeared. Comparison of myocardial levels of reduced pyridine nucleotide fluorescence in hearts treated with leukotriene D4 and in hearts subjected to varying degrees of high-flow hypoxia, or the calcium agonist BAY-K 8644, revealed low levels of reduced pyridine nucleotides in the leukotriene D4-treated hearts, suggesting that leukotriene D4 directly suppressed myocardial contractility. These findings were supported by full restoration of cardiac work by the receptor antagonist FPL 55712 following leukotriene D4 treatment. It is concluded that adenosine and iloprost are potent inhibitors of leukotriene D4-induced reduction in coronary flow in guinea-pig hearts, and that myocardial ischaemia and suppressed cardiac work are prevented during leukotriene D4 study in adenosine or iloprost perfused hearts. Low levels of myocardial-reduced pyridine nucleotides during leukotriene D4 treatment and restoration of cardiac work by FPL 55712 indicate that leukotriene D4 may also have a direct suppressive effect on myocardial contractility.

The effect of a ferric iron complex on isolated rat-liver mitochondria. III. Mechanistic aspects of iron-induced calcium efflux

Addition of iron(III)-gluconate complex to isolated rat liver mitochondria induced a net efflux of Ca 2+ which was not inhibited by ruthenium red. This process resulted in the enhancement of Ca 2+ cycling and a consequent membrane potential drop. Under these experimental conditions the content of mitochondrial glutathione did not appear to be critically modified, whereas an extensive oxidation of mitochondrial pyridine nucleotides was parallelly detected. Iron failed to induce appreciable changes in the oxidation level of pyridine nucleotides in mitochondria isolated from rats fed a selenium deficient diet, a condition in which mitochondrial glutathione peroxidase resulted inhibited by 80%. The iron-induced Ca 2+ release in Se-deficient mitochondria appeared largely delayed and the membrane potential of these mitochondria did not present gross alterations. Iron was also found to induce a transient increase in the mitochondrial cyanide-in-sensitive oxygen consumption. This effect was largely prevented by the addition of the hydrogen peroxide scavenger catalase. It was concluded that iron induced the activation of a specific Ca 2+ efflux pathway via the oxidation of pyridine nucleotides due to the hydrogen peroxide metabolism by glutathione enzyme system.

Blood Pyridine Nucleotide Levels Reflect Niacin Equivalent Intake in Humans

The relationship between the blood NAD and NADP levels and the niacin equivalent intake was investigated using women students. The daily niacin equivalent intake was 151±64μmol (18.5±7.5mg in terms of nicotinamide, mean±SD). The blood NAD and NADP levels were 34.7±7.0 and 19.3±2.3nmol/ml whole blood, respectively. The blood NAD level was proportional to the blood NADP level. Both blood levels were also proportional to the hemoglobin content, and the NAD and NADP levels per g hemoglobin were 233.7±44.7 and 156.3±25.5nmol. The blood NAD and NADP levels increased with an increase in the niacin equivalent intake. From these results, it is concluded that the blood total pyridine nucleotide level (NAD+NADP) reflects the niacin equivalent intake. Accordingly, the blood pyridine nucleotide level may be useful as an index for niacin nutrition in humans.

Pyridine Nucleotides in Selected Plant Species Ecological and Evolutionary Aspects

Summary The pyridine nucleotide levels and reduction charges in selected species of the plant kingdom are described. Data, determined in plants growing under natural conditions (without any treatment) are discussed from physiological and evolutionary aspects. The conclusions are based on the interpretation of data from representative plants of 40 species, using own results of 17 species and data from literature. The results show the existence of special proportions within the pyridine nucleotide system of plants — independent of their evolution level and the metabolic pathway which is mainly responsible for energy supply (ARC > CRC; ARC ≥ 0.5). The NADP(H)/NAD(H) ratio of heterotrophic systems is lower than 1, whereas autotrophic systems show a ratio of 1 or higher. Possibly, NADP(H)/NAD(H) ratios higher than 3 indicate C4-and CAM-NADP-ME types of CO2 fixation. Anaerobic growth leads to an increase in the catabolic reduction charge (CRC ≥ 0.5), whereas a CRC lower than 0.5 is typical of plants under normoxia. A low reduction charge (RC

Relationship between pyridine nucleotide levels and ribonucleotide reductase activity in yoshida ascites hepatoma AH 130

We measured both pyridine nucleotide levels and ribonucleotide reductase-specific activity in Yoshida ascites hepatoma cells as a function of growth in vivo and during recruitment from non-cycling to cycling state in vitro. Oxidized nicotinamide adenine dinucleotide (NAD +) and reduced nicotinamide adenine dinucleotide (NADP) levels remained unchanged during tumour growth, while NADP + and reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels were very high in exponentially growing cells and markedly decreased in the resting phase. Ribonucleotide reductase activity paralleled NADP(H) (NADP + plus NADPH) intracellular content. The concomitant increase in both NADP(H) levels and ribonucleotide reductase activity was also observed during G1-S transition in vitro. Cells treated with hydroxyurea showed a comparable correlation between the pool size of NADP(H) and ribonucleotide reductase activity. On the basis of these findings, we suggest that fluctuations in NADP(H) levels and ribonucleotide reductase activity might play a critical role in cell cycle regulation.

Glutathione cycle activity and pyridine nucleotide levels in oxidant-induced injury of cells.

Exposure of target cells to a bolus of H2O2 induced cell lysis after a latent period of several hours, which was prevented only when the H2O2 was removed within the first 30 min of injury by addition of catalase. This indicated that early metabolic events take place that are important in the fate of the cell exposed to oxidants. In this study, we described two early and independent events of H2O2-induced injury in P388D1 macrophagelike tumor cells: activation of the glutathione cycle and depletion of cellular NAD. Glutathione cycle and hexose monophosphate shunt (HMPS) were activated within seconds after the addition of H2O2. High HMPS activity maintained glutathione that was largely reduced. However, when HMPS activity was inhibited--by glucose depletion or by incubation at 4 degrees C--glutathione remained in the oxidized state. Total pyridine nucleotide levels were diminished when cells were exposed to H2O2, and the breakdown product, nicotinamide, was recovered in the extracellular medium. Intracellular NAD levels fell by 80% within 20 min of exposure of cells to H2O2. The loss of NADP(H) and stimulation of the HMPS could be prevented when the glutathione cycle was inhibited by either blocking glutathione synthesis with buthionine sulfoximine (BSO) or by inhibiting glutathione reductase with (1,3-bis) 2 chlorethyl-1-nitrosourea. The loss of NAD developed independently of glutathione cycle and HMPS activity, as it also occurred in BSO-treated cells.

Activation-associated alterations in neutrophil pyridine nucleotide levels: a potential regulatory role for calcium and calmodulin

The concentration of NADP + NADPH in resting human neutrophils has been measured to be 24.0±2.7·10 −18 mol/cell. Upon activation with opsonized zymosan A, phorbol myristic acetate or N-formylmethionylleucylphenylalanine, neutrophil NADP + NADPH pools increase to 80.5, 84.0, and 54.0·10 −18 mol/cell, respectively. These increases in pyridine nucleotide concentration are blocked by the addition of the calcium antagonist 8-( N, N-dimethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride, while calcium ionophore A23187, in the presence of calcium, will trigger the increase in the absence of other stimuli. Calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide also inhibit stimulus-induced increases in the NADP + NADPH pool. These studies are interpreted as suggesting a role for calcium and calmodulin, and possibly protein kinase C in the regulation of pyridine nucleotide concentration in the activated neutrophil.

Accumulation of Ca2+ induced by cytotoxic levels of menadione in the isolated, perfused rat liver.

Previous studies have indicated that the presence of cytotoxic levels of menadione (2-methyl-1,4-naphthoquinone) causes rapid changes in intracellular thiol and Ca2+ homeostasis in isolated rat hepatocytes. The present investigation was undertaken to examine these effects in the intact liver. Rat livers were therefore perfused with Krebs-Henseleit buffer containing 1.3 mM Ca2+ using a single-pass mode, and the perfusate Ca2+ level was monitored with an on-line Ca2+-selective electrode. Infusion of menadione elicited an increased O2 uptake by the liver, followed by a dose-dependent decrease in the perfusate level of Ca2+. Hepatic accumulation of Ca2+ was accompanied by stimulation of cytosolic phosphorylase a activity. Cessation of menadione infusion resulted in gradual recovery of perfusate Ca2+ to base levels. Ca2+ uptake was not accompanied by decreases in reduced pyridine nucleotide or ATP levels in the liver as evidenced by measurements either during maximal Ca2+ uptake or after recovery. However, Ca2+ uptake was correlated with decreased glutathione and increased glutathione disulfide levels in the liver, both of which reversed during recovery from Ca2+ uptake. Moreover, depletion of hepatic glutathione by pretreatment with diethylmaleate resulted in increased Ca2+ uptake during menadione infusion. The amount of protein-bound mixed disulfides showed a particularly striking relationship to Ca2+ uptake, reaching a maximal level during Ca2+ uptake and reversing toward normal value during recovery from Ca2+ accumulation. The present findings suggest that menadione-induced Ca2+ uptake is due to plasma membrane dysfunction as a result of loss of protein thiol groups critical for maintaining the plasma membrane Ca2+ extrusion mechanism. Our model offers a particularly useful opportunity to study mechanisms underlying toxic disturbances in Ca2+ homeostasis in the intact liver, since Ca2+ fluxes can be monitored under conditions in which cellular control mechanisms are not obliterated by excessive toxicity.