Glutamate-induced differential mitochondrial response in young and adult rats

Excitatory amino acid glutamate is involved in neurotransmission in the nervous system but it becomes a potent neurotoxin under variety of conditions. However, the molecular mechanism of excitotoxicity is not known completely. We have studied the influence of glutamate on intracellular calcium and mitochondrial functions in cortical slices from young and adult rats. The slices from both the age groups exhibited comparable intracellular calcium changes upon glutamate stimulation. Glutamate treatment caused a decrease in adenosine 5'-diphosphate/adenosine 5'-triphosphate (ADP/ATP) and an increase in nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide reduced form (NAD/NADH) ratio in both the age groups but the magnitude and the nature of temporal change was different. Glutamate-induced decrease in ATP/ADP and increase in NAD/NADH ratio was significantly higher in slices from the adult as compared to the young rats. The slices from young rats elicited slightly higher mitochondrial depolarization than adult rats. However, the formation of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) release were significantly higher in adult rats as compared to young rats. Glutamate-induced mitochondrial depolarization, ROS formation and LDH release were highly dependent on the presence of Ca(2+) in the extracellular medium. The treatment of slices with mitochondrial inhibitors rotenone and oligomycin inhibited ROS formation and LDH release substantially. Our results suggest that the glutamate-induced increase in intracellular calcium is not the only factor responsible for neuronal cell death but the mitochondrial functions could be crucial in excitotoxicity.     

Probing glycolytic and membrane potential oscillations in Saccharomyces cerevisiae

We have investigated glycolytic oscillations under semi-anaerobic conditions in Saccharomyces cerevisiae by means of NADH fluorescence, measurements of intracellular glucose concentration, and mitochondrial membrane potential. The glucose concentration was measured using an optical nanosensor, while mitochondrial membrane potential was measured using the fluorescent dye DiOC 2(3). The results show that, as opposed to NADH and other intermediates in glycolysis, intracellular glucose is not oscillating. Furthermore, oscillations in NADH and membrane potential are inhibited by the ATP/ADP antiporter inhibitor atractyloside and high concentrations of the ATPase inhibitor N, N'-dicyclohexylcarbodiimide, suggesting that there is a strong coupling between oscillations in mitochondrial membrane potential and oscillations in NADH mediated by the ATP/ADP antiporter and possibly also other respiratory components.     

Study of the regulation of oxidation and CO2 assimilation in intact Nitrobacter winogradskyi cells.

1. Changes of the adenine nucleotides in resting and growing Nitrobacter winogradskyi cells were measured in connection with regulating processes during nitrite oxidation and endogenous respiration. 2. After the addition of nitrite to endogenously respiring cells the ATP pool increased strongly during the first 60 sec at the expense of the ADP pool. At this point the energy charge was approx. 0.55. After the first 90 sec the ATP pool dropped, oscillating, to a lower level. The CO2 assimilation began at this point. 3. Under a nitrogen atmosphere the AMP pool increased and the ATP pool decreased. With a value of approx. 0.17 the energy charge was extremely low. When oxygen was added the Nitrobacter cells began to oxidize stored NADH. The ATP pool increased in a few seconds whereas the AMP pool decreased. The P/O ratio of endogenously respiring cells equaled 0.6 under these conditions. 4. During the changeover from anaerobic to aerobic conditions and in the presence of nitrite the nitrite oxidation and CO2 assimilation, opposed to aerobic conditions, were inhibited at first after the nitrite addition. The changeover of the respiratory chain enzymes from a reduced to an oxidized charge and the ATP increase were delayed in comparison with experiments without nitrite. According to these findings the endogenous respiration must be almost nil while nitrite oxidizing cells are growing.     

H(2)O(2) modulates purinergic-dependent calcium signalling in osteoblast-like cells

Reactive oxygen species (ROS) have long been considered as toxic by-products of aerobic metabolism and appear involved in the pathogenesis of degenerative diseases. The physiological role of ROS as second messengers in cell signal transduction is, on the other hand, increasingly recognized. Here we investigated the effects of H(2)O(2) and extracellular nucleotides on calcium signalling in four osteoblastic cell lines. In the highly differentiated HOBIT cells, sensitive to nanomolar concentrations of ADP and UTP, millimolar H(2)O(2) induced oscillatory increases of the cytosolic calcium concentration followed by a steady and sustained calcium increase. Long lasting rhythmic calcium activity was induced by micromolar H(2)O(2) doses. The H(2)O(2)-induced calcium signals, due to both release from intracellular stores and influx from the extracellular milieu, were totally prevented by incubating the cells with the P2 receptor antagonist suramin or with the ATP/ADP hydrolyzing enzyme apyrase. In the osteosarcoma SaOS-2 cells micromolar H(2)O(2) failed to evoke calcium signals and millimolar H(2)O(2) induced a slowly developing calcium influx which was unaffected by suramin and apyrase. These cells responded to micromolar concentrations of ATP and ADP, but were largely insensitive to UTP. ROS 17/2.8 osteosarcoma cells were totally insensitive to ATP, ADP and UTP in keeping with the evidence that these cells lack functional purinergic receptors. In these cells, H(2)O(2) up to 1mM did not increase the cytosolic calcium concentration. In ROS/P2Y(2) cells, stably expressing the P2Y(2) receptor, spontaneous calcium oscillations were observed in 38% of the population and nanomolar concentration of extracellular ATP or UTP activated oscillations in quiescent cells. Spontaneous calcium signals were inhibited by suramin and apyrase. In these cells H(2)O(2) induced oscillatory calcium activity that was blocked by suramin and apyrase. The sensitivity of ROS/P2Y(2) cells to UTP decreased significantly in the presence of DTT, which was effective also in inhibiting spontaneous calcium oscillations. On the other hand, the membrane-impermeant thiol oxidant DTNB induced calcium oscillations that were inhibited by incubating the cells with suramin or apyrase. Since peroxide did not increase extracellular ATP in these cell lines, we propose that, in osteoblasts, mild oxidative conditions could activate purinergic signalling through the sensitization of P2Y(2) receptor.     

Energy-dependent formation of free ATP in yeast submitochondrial particles, and its stimulation by oligomycin

Yeast submitochondrial particles, in a P_i- and NADH-dependent reaction, produced low concentrations of free ATP in the absence of added ADP. This formation of free ATP, as measured by the luciferin-luciferase method, was strongly stimulated by oligomycin. For maximal stimulation, oligomycin was to be added not earlier than 5–10 min after the addition of NADH. Upon addition of antimycin or FCCP the system was completely inhibited. The amount of free ATP formed corresponded to one-third of the amount of bound ATP in submitochondrial particles. The stimulatory effect of oligomycin disappeared if the submitochondrial particles were spun down after oligomycin stimulation and then resuspended in the reaction medium, whereas submitochondrial particles with no oligomycin added initially were stimulated by oligomycin after the same procedure. A different picture emerged with addition of ADP. If the submitochondrial particles were preenergized with NADH in the presence of oligomycin before the addition of ADP the formation of free ATP upon subsequent addition of ADP was inhibited by oligomycin. In the presence of oligomycin, but lacking preenergization with NADH, a stimulation of free ATP formation was achieved with added ADP. A possible explanation for the stimulating effect of oligomycin on ATP formation in the absence of added ADP is that it enhances the release of bound ATP in an energy-requiring process. The release of only about one-third of the bound ATP could indicate that one of three nucleotide-binding subunits involved in the mechanism of ATP formation by ATP synthase is in a state suitable for such an energy-dependent release of ATP.     

Methodological problems of direct bioluminescent ADP assay in platelets and erythrocytes

We have developed a method for ADP bioluminescent measurement in platelets and erythrocytes which complements our previous method for ATP assay. When the different parameters of the system under investigation are taken into account, a linea range between 10(-9) and 10(-7) g/ml can be obtained without incubation or troublesome extraction. This makes the method easy and useful for identifying any disease-induced alterations in ATP and/or ADP levels in these blood cells. The data obtained correlate well with those of a bioluminescent method requiring extraction with ethanol/EDTA and incubation, giving the reference intervals of 3.5-5.5 mumol/10(11) PLT for ATP determination and 1.9-3.7 mumol/10(11) PLT for ADP determination in platelets, and 3.2-3.8 mumol/g Hgb for ATP determination and 0.56-0.73 mumol/g Hgb for ADP in erythrocytes. This assay was applied to quality control on blood bags in transfusion centers and proved to be a rapid and reliable method for testing the viability of stored blood cells.     

Measurement of adenine nucleotides in plasma

The goal of this study was to identify the most important variables affecting bioluminescent ATP, ADP and AMP measurements in plasma and to develop an assay that takes these variables into account. Blood samples were drawn from conscious dogs. A ‘stop solution’ containing EDTA was prepared, which greatly retarded plasma ATP degradation by chelating Mg⁺² and Ca⁺² that are co‐factors for many ATPases. Stop solution and blood were mixed using a two‐syringe withdrawal system. Samples were centrifuged twice in order to remove red blood cells, and ATP was measured in the supernatant using the firefly luciferase assay. Sample pH was adjusted to the optimal range (7.75–7.95) and Mg²⁺ (necessary for the luciferase reaction) was added back to the sample within the luminometer 2 s prior to luciferase addition. Four assay tubes were prepared for each plasma sample, containing standard additions of 0–15 pmol added ATP, in order to quantify native plasma ATP content. In separate plasma/stop solution samples ADP + ATP was measured after converting ADP to ATP via the pyruvate kinase reaction, and AMP + ADP + ATP was measured after addition of both myokinase and pyruvate kinase. Addition of forskolin and isobutylmethylxanthine (IBMX) to the stop solution to inhibit platelets resulted in lower ATP concentrations. Measurement of ATP and haemoglobin from lysed erythrocytes revealed that haemolysis exerts a strong influence on plasma ATP concentration that must be taken into account. Copyright © 2003 John Wiley & Sons, Ltd.     

Deep Origin of Plastid/Parasite ATP/ADP Translocases

Abstract Membrane proteins that transport ATP and ADP have been identified in mitochondria, plastids, and obligate intracellular parasites. The mitochondrial ATP/ADP transporters are derived from a broad-specificity transport family of eukaryotic origin, whereas the origin of the plastid/parasite ATP/ADP translocase is more elusive. Here we present the sequences of five genes coding for ATP/ADP translocases from four species of Rickettsia. The results are consistent with an early duplication and divergence of the five ATP/ADP translocases within the rickettsial lineage. A comparison of the phylogenetic depths of the mitochondrial and the plastid/parasite ATP/ADP translocases indicates a deep origin for both transporters. The results provide no evidence for a recent acquisition of the ATP/ADP transporters in Rickettsia via horizontal gene transfer, as previously suggested. A possible function of the two types of ATP/ADP translocases was to allow switches between glycolysis and aerobic respiration in the early eukaryotic cell and its endosymbiont.     

Ectoadenylate kinase and plasma membrane ATP synthase activities of human vascular endothelial cells

Formation of ATP from ADP on the external surface of vascular endothelial cells has been attributed to plasma membrane ATP synthase, ectoadenylate kinase (ecto-AK), and/or ectonucleoside diphosphokinase. These enzymes or their catalytic products have been causatively linked to the elaboration of vascular networks and the regulation of capillary function. The amount of ATP generated extracellularly is small, requiring sensitive analytical methods for quantification. Human umbilical vein endothelial cells were used to revisit extracellular ATP synthesis using a reliable tetrazolium reduction assay and multiwell plate cultures. Test conditions compatible with AK stability were established. Extracellular AK activity was found to be <1% of the total (intracellular and extracellular), raising the possibility that the external enzyme could have leaked from living cells and/or a few dying cells. To determine whether AK inadvertently leaked from the cells, the activity of another cytoplasmic enzyme, glucose-6-phosphate dehydrogenase (G6PD), was also measured. G6PD is present in the cytoplasm in similar abundance to AK. The activity ratio of G6PD (extracellular/total) was found to be similar to that of AK. Because G6PD in the medium was probably due to leakage, other cytoplasmic macromolecules, including AK, should be released proportionately from the cells. The role of plasma membrane ATP synthase in extracellular ATP formation was examined using Hanks' balanced salt solution with and without selective inhibitors of AK and ATP synthase activities. With P(1),P(5)-di(adenosine 5')-pentaphosphate (inhibitor of AK activity), no extracellular ATP synthesis was detected, whereas with oligomycin, piceatannol, and aurovertin (inhibitors of F(1)F(0)-ATP synthase and F(1)-ATPase activities), no inhibition of extracellular ATP synthesis was observed. AK activity alone could account for the observed extracellular ATP synthesis. The possible impact of ADP impurity in the assays is discussed.     

Studies on the effects of coenzyme A-SH: acetyl coenzyme A, nicotinamide adenine dinucleotide: reduced nicotinamide adenine dinucleotide, and adenosine diphosphate: adenosine triphosphate ratios on the interconversion of active and inactive pyruvate dehydrogenase in isolated rat heart mitochondria.

The content of coenzyme A-SH (CoASH) and acetyl-CoA of suspensions of rat heart mitochondria was stabilized by the addition of DL-carnitine and acetyl-DL-carnitine, in the presence of the respiratory inhibitor rotenone. The mitochondrial content of NAD+ and NADH was similarly stabilized by the addition of acetoacetate and DL-3-hydroxybutyrate, and the content of ADP and ATP was imposed by the addition of these nucleotides to the mitochondrial suspension, in the presence of uncoupling agent and oligomycin, to inhibit ATPase. Under these conditions, mitochondrial CoASH/acetyl-CoA, NAD+/ NADH, and ADP/ATP ratios could be varied independently, and the effect on the interconversion of active and inactive pyruvate dehydrogenase could be studied. Decreases in both CoASH/acetyl-CoA and NAD+/NADH ratios were shown to be inhibitory to the steady state activity of pyruvate dehydrogenase, and this effect is described at three different ADP/ATP ratios and different concentrations of added MgCl2. A new steady state level of activity was achieved within 10 min of a change in either CoASH/acetyl-CoA or NAD+/NADH ratio; the rate of inactivation was much higher than the rate of reactivation under these conditions. Effects of CoASH/acetyl-CoA and NAD+/NADH may be additive but are still quantitatively lesser than the changes in activity of pyruvate dehydrogenase induced by changes in ADP/ATP ratio. The variation in activity of pyruvate dehydrogenase with ADP/ATP ratio is described in the absence of changes in the other two ratios, conditions which were not met in earlier studies which employed the oxidation of different substrates to generate changes in all three ratios.     

Increased levels of ATP and NADH are associated with increased solvent production in continuous cultures of Clostridium acetobutylicum

Summary Levels of intracellular NAD, NADH, ADP, and ATP were measured in batch and continuous cultures of Clostridium acetobutylicum. ATP levels during glucose-sufficient (non-glucose limited), solvent-producing steady states were five to eight times as high as in glucose-limited (acidogenic) steady state cultures (4.8 mole/g versus 0.9 mole/g). NADH was also higher in glucose-sufficient cultures, although the increase was not as pronounced as with ATP. When glucose-limited cultures were sparged with CO, the resulting shift to solvent production was accompanied by three- or four-fold increases in NADH and ATP levels. In batch cultures, NADH and ATP levels were relatively high at all times compared to levels in continuous cultures. Although the levels of these nucleotides showed systematic trends during normal batch fermentations, there was no significant change with the onset of solvent production.     

KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion

Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in β-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS–1 cells. Taking advantage of hemicannels’opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 μM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 μM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of K_ATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.     

Growth inhibition of transformed mouse fibroblasts by adenine nucleotides occurs via generation of extracellular adenosine

The growth of transformed mouse fibroblasts (3T6 cells) in medium containing 5% fetal bovine serum was inhibited after treatment with concentrations greater than 50 microM ATP, ADP, or AMP. Adenosine, the common catabolite of the nucleotides, had no effect on cell growth at concentrations below 1 mM. However, the following results indicate that the toxicity of ATP, ADP, and AMP is mediated by serum- and cell-associated hydrolysis of the nucleotides to adenosine. 1) ADP and AMP, but not ATP, were toxic to 3T6 cells grown in serum-free medium or medium in which phosphohydrolase activity of serum was inactivated. Under these conditions, the cells exhibited cell-associated ADPase and 5'-nucleotidase activity, but little ecto-ATPase activity. 2) Inhibition of adenosine transport in 3T6 cells by dipyridamole or S-(p-nitrobenzyl)-6-thioinosine prevented the toxicity of ATP in serum-containing medium and of ADP and AMP in serum-free medium. 3) A 16-24-h exposure to 125 microM AMP or ATP was needed to inhibit cell growth under conditions where serum- and cell-associated hydrolysis of the nucleotides generated adenosine in the medium continuously over the same time period. In contrast, 125 microM adenosine was completely degraded to inosine and hypoxanthine within 8-10 h. Furthermore, multiple doses of adenosine added to the cells at regular intervals over a 16-h period were significantly more toxic than an equivalent amount of adenosine added in one dose. Treatment of 3T6 cells with AMP elevated intracellular ATP and ADP levels and reduced intracellular UTP levels, effects which were inhibited by extracellular uridine. Uridine also prevented growth inhibition by ATP, ADP, and AMP. These and other results indicate that serum- and cell-associated hydrolysis of adenine nucleotides to adenosine suppresses growth by adenosine-dependent pyrimidine starvation.     

Energy-dependent formation of free ATP in yeast submitochondrial particles, and its stimulation by oligomycin

Yeast submitochondrial particles, in a Pi- and NADH-dependent reaction, produced low concentrations of free ATP in the absence of added ADP. This formation of free ATP, as measured by the luciferin-luciferase method, was strongly stimulated by oligomycin. For maximal stimulation, oligomycin was to be added not earlier than 5-10 min after the addition of NADH. Upon addition of antimycin or FCCP the system was completely inhibited. The amount of free ATP formed corresponded to one-third of the amount of bound ATP in submitochondrial particles. The stimulatory effect of oligomycin disappeared if the submitochondrial particles were spun down after oligomycin stimulation and then resuspended in the reaction medium, whereas submitochondrial particles with no oligomycin added initially were stimulated by oligomycin after the same procedure. A different picture emerged with addition of ADP. If the submitochondrial particles were preenergized with NADH in the presence of oligomycin before the addition of ADP the formation of free ATP upon subsequent addition of ADP was inhibited by oligomycin. In the presence of oligomycin, but lacking preenergization with NADH, a stimulation of free ATP formation was achieved with added ADP. A possible explanation for the stimulating effect of oligomycin on ATP formation in the absence of added ADP is that it enhances the release of bound ATP in an energy-requiring process. The release of only about one-third of the bound ATP could indicate that one of three nucleotide-binding subunits involved in the mechanism of ATP formation by ATP synthase is in a state suitable for such an energy-dependent release of ATP.     

Catabolism of Ap4A and Ap3A in whole blood. The dinucleotides are long-lived signal molecules in the blood ending up as intracellular ATP in the erythrocytes

Adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')triphospho(5')adenosine (Ap3A) are stored in large amounts in human platelets. After activation of the platelets both dinucleotides are released into the extracellular milieu where they play a role in the modulation of platelet aggregation and also in the regulation of the vasotone. It has recently been shown that the dinucleotides are degraded by enzymes present in the plasma [Lüthje, J. & Ogilvie, A. (1987) Eur. J. Biochem. 169, 385-388]. The further metabolism as well as the role of blood cells has not been established. The dinucleotides were first degraded by plasma phosphodiesterases yielding ATP (ADP) plus AMP as products which were then metabolized to adenosine and inosine. The nucleosides did not accumulate but were very rapidly salvaged by erythrocytes yielding intracellular ATP as the main product. Although lysates of platelets, leucocytes and red blood cells contained large amounts of Ap3A-degrading and Ap4A-degrading activities, these activities were not detectable in suspensions of intact cells suggesting the lack of dinucleotide-hydrolyzing ectoenzymes. Compared to ATP, which is rapidly degraded by ectoenzymes present on blood cells, the half-life of Ap4A was two to three times longer. Since the dinucleotides are secreted together with ADP and ATP from the platelets, we tested the influence of ATP on the rate of degradation of Ap4A. ATP at concentrations present during platelet aggregation strongly inhibited the degradation of Ap4A in whole blood. It is suggested that in vivo the dinucleotides are protected from degradation immediately after their release. They may thus survive for rather long times and may act as signals even at sites far away from the platelet aggregate.     

Adenine and pyridine nucleotides in the erythrocyte of different mammalian species

Adenine (ATP, ADP, AMP) and pyridine nucleotides (NADP+, NADPH, NAD+, NADH) concentrations have been determined by HPLC in the erythrocytes from five different mammalian species (pig, rat, mouse, rabbit and cow) and compared to those in human red blood cells. Two different extraction procedures have been used and the results obtained are compared and discussed. A good correlation between the different abilities of the erythrocytes of the six species to utilize glucose and the NAD+/NADH ratio was found, with high NAD+/NADH ratio in the red blood cell of the species with high glucose utilization rates. The levels of all the glycolytic enzymes and some of the pentose phosphate shunt enzymes were also determined.     

Bacterial DNA supercoiling and [ATP]/[ADP]. Changes associated with a transition to anaerobic growth.

Shifting Escherichia coli from aerobic to anaerobic growth caused changes in the ratio of [ATP]/[ADP] and in negative supercoiling of chromosomal and plasmid DNA. Shortly after lowering oxygen tension, both [ATP]/[ADP] and supercoiling transiently decreased. Under conditions of exponential anaerobic growth, both were higher than under aerobic conditions. These correlations may reflect an effect of [ATP]/[ADP] on DNA gyrase, since in vitro [ATP]/[ADP] influences the level of plasmid supercoiling attained when gyrase is either introducing or removing supercoils. When the supercoiling activity of gyrase was perturbed by a mutation in gyrB, a shift to anaerobic conditions resulted in plasmid supercoil relaxation similar to that seen with wild-type. However, the low level of supercoiling in the mutant persisted during a time when supercoiling in wild-type recovered and then exceeded aerobic levels. Thus, changes in oxygen tension can alter DNA supercoiling through an effect on gyrase, and correlations exist between changes in supercoiling and changes in the intracellular ratio of [ATP]/[ADP].     

Extracellular adenine compounds, red blood cells and haemostasis: facts and hypotheses

Previously, the role of adenine nucleotides was thought to be confined to the intracellular space of the cell. Research of the last decades has revealed that nucleotides also occur in the extracellular milieu. This survey deals with extracellular adenine compounds in the blood, focussing on their role as chemical mediators in the haemostatic effect of red cells. Erythrocytes may act as pro-aggregatory cells by at least two chemical mechanisms. Firstly, they can enhance platelet aggregation by releasing adenosine diphosphate (ADP), a well known platelet stimulatory substance. ADP is set free when red cells are stressed mechanically, for instance by shear forces generated in the blood stream; ample experimental evidence supporting this view is summarized. Secondly, erythrocytes efficiently take up extracellular adenosine via their nucleoside transporters, thereby removing a potent inhibitor of platelet function. Extracellular adenosine occurs in the blood stream, either directly released from various tissues or as the end product of extracellular adenine nucleotide metabolism, e.g. after degradation of red cell-born ADP or ATP. Finally, a novel mechanism of action of the antithrombotic drug dipyridamole, which has very recently been put forward, is demonstrated. Dipyridamole inhibits platelet function indirectly by blocking the uptake of extracellular adenosine via the nucleoside transporter of red cells; increased adenosine levels in turn are responsible for the antiaggregatory effect of dipyridamole.     

Energy metabolism of autotrophically and heterotrophically grown cells of Nitrobacter winogradskyi

The adenine nucleotide pools and the NADH pool were compared in intact Nitrobacter winogradskyi cells grown under different conditions. The NADH pool was highest in nitrite-grown cells (22.0 nmol/mg N), less high in acetategrown cells (15.1 nmol/mg N),and lowest in pyruvate-grown cells (11.9 nmol/mg N).The adenine nucleotide pools and the NADH pool were determined after the transition from anaerobic to aerobic conditions.In both autotrophically and heterotrophically grown cells the ATP pool decreased within the first second after the addition of oxygen and then increased.In cells grown with nitrite or acetate the NADH pool increased the first second after the addition of oxygen then decreased below the initial value. In pyruvate-grown cells the changes in the NADH pool were less obvious.In the presence of rotenone autotrophic cells were able to generate ATP, but the reverse energy-dependent electron transport was inhibited. Consequently, NADH was not synthesized. N,N-dicyclohexylcarbodiimide an inhibitor of ATPase, prevented both ATP and NADH generation.Abbreviations DCCD N,N-dicyclohexylcarbodiimide     

Extracellular ATP stimulates increases in Na+/K+ pump activity, intracellular pH and uridine uptake in cultures of mammalian cells.

Using 3T3 and 3T6 mouse fibroblasts and A431 epidermoid carcinoma cells, we previously observed that extracellular ATP and ADP were mitogens and they synergized with other growth factors (Huang, N., Wang, D. and Heppel, L. A. (1989) Proc. Natl. Acad. Sci. USA 86, 7904-7908). We now report that ATP and ADP stimulated Na+ entry, intracellular alkalinization and Na+/K+ pump activity, which are early events that had been proposed to play a central role in DNA synthesis. In addition, ATP, ADP and AMPPNP stimulated uridine uptake by a pathway involving arachidonic acid metabolism. In A431 cells, activation of protein kinase C also contributed to ATP-dependent stimulation of uridine uptake. Concentrations of indomethacin and pertussis toxin which inhibited uridine uptake also blocked arachidonic acid metabolism and DNA synthesis. ATP acted as a competence factor. Interestingly, ATP did not have to be continuously present to stimulate uridine uptake. It was equally effective even when it was washed away after brief treatment of cells.