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.     

A separation method for the assay of adenylylcyclase, intracellular cyclic AMP, and cyclic-AMP phosphodiesterase using tritium-labeled substrates.

A method for the separation of cyclic AMP from adenosine and polyvalent adenine nucleotides is described. The method consists of the sequential elution of adenosine and cyclic AMP from a single column of acidic aluminum oxide (alumina) with dilute hydrochloric acid and ammonium acetate. Adenosine, adenine, xanthine, and hypoxanthine are rapidly eluted with the application of 0.005 N hydrochloric acid while cyclic AMP remains adsorbed to the alumina. A subsequent application of 0.1 M ammonium acetate elutes more than 90% of the cyclic AMP. Under these conditions, polyvalent nucleotides (AMP, ADP, and ATP) remain adsorbed to the alumina. The method permits the measurement of adenylylcyclase activity using [3H]ATP as the labeled substrate. The same technique can be used to measure the accumulation of cyclic AMP in intact cells after labeling the ATP pool with [3H]adenine. With slight modification, the technique can be used to measure the activity of cyclic-AMP phosphodiesterase using [3H]cyclic AMP as the substrate. The proposed technique provides rapid, highly reproducible assays using inexpensive, disposable columns.     

Uptake of AMP, ADP, and ATP in Escherichia coli W

The uptake activity ratio for AMP, ADP, and ATP in mutant (T-1) cells of Escherichia coli W, deficient in de novo purine biosynthesis at a point between IMP and 5-aminoimidazole-4-carboxiamide-1-β-D-ribofuranoside (AICAR), was 1:0.43:0.19. This ratio was approximately equal to the 5'-nucleotidase activity ratio in E. coli W cells. The order of inhibitory effect on [2-3H]ADP uptake by T-1 cells was adenine > adenosine > AMP > ATP. About 2-fold more radioactive purine bases than purine nucleosides were detected in the cytoplasm after 5 min in an experiment with [8-1?C]AMP and T-1 cells. Uptake of [2-3H]adenosine in T-1 cells was inhibited by inosine, but not in mutant (Ad-3) cells of E. coli W, which lacked adenosine deaminase and adenylosuccinate lyase. These experiments suggest that AMP, ADP, and ATP are converted mainly to adenine and hypoxanthine via adenosine and inosine before uptake into the cytoplasm by E. coli W cells.     

In vitro effect of homocysteine on nucleotide hydrolysis by blood serum from adult rats

During the past few years, elevated blood levels of homocysteine (Hcy) have been linked to increased risk of premature coronary artery disease, stroke and thromboembolism. These processes can be also related to the ratio adenine nucleotide/adenosine, since extracellularly these nucleotides are associated with modulation of processes such as platelet aggregation, vasodilatation and coronary flow. Furthermore, there are some studies that suggest a relationship between Hcy and plasma adenosine concentrations. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes one of the systems for rapid inactivation of circulating adenine nucleotides. Thus, the main objective of this study was to evaluate if Hcy can participate in the modulation of the extracellular adenine nucleotide hydrolysis by rat blood serum. Our results showed that Hcy, at final concentrations of 5.0 mM, inhibits in vitro ATP, ADP and AMP hydrolysis by 26, 21 and 16%, respectively. Also Hcy, at final concentrations of 8.0mM, inhibited the in vitro hydrolysis of ATP, ADP and AMP by 46, 44 and 44%, respectively. Kinetic analysis showed that the inhibitions of the three adenine nucleotide hydrolyses in the presence of Hcy, by serum of adult rats, is of the uncompetitive type. The IC50 calculated from the results obtained were 6.52+/-1.75 mM (n = 4), 5.18 +/- 0.64 mM (n = 3) and 5.16 +/- 1.22 mM (n = 3) for ATP, ADP and AMP hydrolysis, respectively.     

Evidence for two adenine derivative receptors in rat ileum which are not involved in the nonadrenergic, noncholinergic response.

The receptors mediating inhibition of the rat ileum by adenosine and adenine nucleotides were studied. ATP and ADP were more potent than AMP or adeonsine. Theophylline antagonized the effects of adenosine and AMP but not those of ATP or ADP. Preparations desensitized to ATP or ADP were still inhibited by adenosine and vice versa. The nonadrenergic, noncholinergic inhibition produced by field stimulation or nicotine was not attenuated by the presence of theophylline or desensitization to ATP. These data indicate that more than one adenine derivative receptor is present in rat ileum and that ATP and adenosine are unlikely candidates for the unknown transmitter.     

Regulation of human neutrophil functions by adenine nucleotides

Previous work has shown that platelet-derived adenine nucleotides modulate neutrophil superoxide anion (O2-) generation. Additional studies were undertaken to characterize the effects of authentic adenosine (ADO) and its nucleotide derivatives on the inflammatory functions of human neutrophils. Stimulus-specific inhibition of neutrophil O2- generation by ADO in response to FMLP was verified. In addition, the ability of ATP, ADP, and AMP to limit neutrophil O2- generation induced by FMLP (0.2 to 0.5 microM) was demonstrated. The concentration producing 50% inhibition for nucleotide inhibition of neutrophil O2- generation was in the rank order of ADO (0.1 microM) less than AMP (0.5 microM) less than ADP less than or equal to ATP (5 microM). Guanine and inosine nucleotides (0.01 to 100 microM) did not inhibit FMLP-stimulated neutrophil O2- generation. Neutrophil degranulation in response to FMLP was only modestly inhibited by adenine nucleotides and ADO. Adenosine and ADP failed to affect chemotaxis of neutrophils stimulated with FMLP. The inability of non-metabolizable analogs to mimic the inhibitory effects of authentic ATP or ADP on the neutrophil O2- response suggested that metabolism of added nucleotides is necessary for their effectiveness. Both TLC and HPLC confirmed that ATP and ADP were converted to AMP and ADO after their incubation with unstimulated or FMLP-activated neutrophils. The addition of adenosine deaminase to neutrophil reaction mixtures in which conversion of added nucleotides was apparent removed detectable ADO but failed to completely abrogate the inhibition of neutrophil O2- generation by accumulated AMP. The kinetics of inhibition of FMLP-induced neutrophil O2- generation by ATP and ADP also indicated that conversion of these nucleotides to ADO and/or AMP may be essential for their ability to reduce neutrophil responses.     

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase

Adenosie, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phophodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell suface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides has no effect on the accumulation of cyclic AMP. Among other adenine nucleotides was tested, adenosine 5′-monophosphoramidate, but not adenosine 5′-monosulfate, significantly increased cyclic AMP especially with the addition of papaverine. Neither 2′- nor 3′-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Adenylate kinase of plants. Properties of adenylate kinase of pea leaves]

The properties of adenylate kinase in 2 ADP in equilibrium ATP + AMP reaction have been studied. The dependence of the enzyme activity on medium pH, protein concentration, substrates, Mg++ ions, AMP, adenine and adenosine has been also investigated. pH optimum is found to be 8.5 for forward reaction and 8-9--for the reverse one. The Michaelis constants are as follows: for ADP--1.17-10(-4) M, for ATP--3.33-10(-4) M at 24 degrees C, in 50 mM tris-HCl pH 7.6. The optimal ratio, Mg++ ions/substrates (ADP, ATP + AMP), is 1:2. The chelates of adenine nucleotides with Mg++ ions are proved to be "true" reaction substrates. Unlike adenine and adenosine, the product of AMP reaction inhibits adenylate kinase activity. It is concluded that the properties of adenylate kinase in plants are similar to those of animals and humans (moikinase).     

Role of membrane-bound 5''-nucleotidase in nucleotide uptake by the moderate halophile Vibrio costicola

Intact cells of Vibrio costicola hydrolyzed ATP, ADP, and AMP. The membrane-bound 5'-nucleotidase (C. Bengis-Garber and D. J. Kushner, J. Bacteriol. 146:24-32, 1981) was solely responsible for these activities, as shown by experiments with anti-5'-nucleotidase serum and with the ATP analog, adenosine 5'-(beta gamma-imido)-diphosphate. Fresh cell suspensions rapidly accumulated 8-14C-labeled adenine 5'-nucleotides and adenosine. The uptake of ATP, ADP, and AMP (but not the adenosine uptake) was inhibited by adenosine 5'-(beta gamma-imido)-diphosphate similarly to the inhibition of the 5'-nucleotidase. Furthermore, the uptake of nucleotides had Mg2+ requirements similar to those of the 5'-nucleotidase. The uptake of ATP was competitively inhibited by unlabeled adenosine and vice versa; inhibition of the adenosine uptake by ATP occurred only in the presence of Mg2+. These experiments indicated that nucleotides were dephosphorylated to adenosine before uptake. The hydrolysis of [alpha-32P]ATP as well as the uptake of free adenosine followed Michaelis-Menten kinetics. The kinetics of uptake of ATP, ADP, and AMP also each appeared to be a saturable carrier-mediated transport. The kinetic properties of the uptake of ATP were compared with those of the ATP hydrolysis and the uptake of adenosine. It was concluded that the adenosine moiety of ATP was taken up via a specific adenosine transport system after dephosphorylation by the 5'-nucleotidase.     

A new chromatographic method using immobilized acriflavin for measuring cyclic AMP in cells prelabeled with radioactive adenine.

A method using the principle of charge-transfer chromatography has been developed for the determination of cyclic AMP levels in intact prelabeled cells. The ATP pool was prelabeled by incubating the cells in the presence of radioactive adenine. The cyclic AMP formed from ATP was extracted with HC10(4) and separated from adenine and other adenosine-related nucleotides by chromatography on acriflavin-Sephadex G-25. This method provides a rapid and sensitive isolation of cyclic AMP with high recovery (95-100%) and low blnks. Further, no contamination of the cyclic AMP fractions was found by either adenine or adenosine nucleotides such as ATP, ADP or AMP. This procedure is applicable to a variety of cell or tissue systems.     

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase.

Adenosine, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phosphodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell surface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides had no effect on the accumulation of cyclic AMP. Among other adenine nucleotides we tested, adenosine 5'-monophosphoramidate, but not adenosine 5'-monosulfate significantly increased cyclic AMP especially with the addition of papaverine. Neither 2'- nor 3'-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Measurements of ATP in mammalian cells

Levels of phosphorylated adenosine nucleotides, including the universal energy carrier adenosine 5(')-triphosphate (ATP) and its metabolites adenosine 5(')-diphosphate (ADP) and adenosine 5(')-monophosphate (AMP), define the energy state in living cells and are dependent mainly on mitochondrial function. In this article, we describe a method based on the luciferase-luciferin system used to measure mitochondrial ATP synthesis continuously in permeabilized mammalian cells and mitochondria isolated from animal tissues. We also describe a technique that uses the expression of recombinant targeted luciferase to report ATP content in different cell compartments. Finally, we describe an HPLC-based method for accurate measurement of ATP, ADP, and AMP in cultured cells and animal tissues.     

Transport of AMP by Rickettsia prowazekii.

Rickettsia prowazekii possesses an exchange transport system for AMP. Chromatographic analysis of the rickettsiae demonstrated that transported AMP appeared intracellularly as AMP, ADP, and ATP, and no hydrolytic products appeared in either the intracellular or extracellular compartments. The phosphorylation of AMP to ADP and ATP was prevented by pretreatment of the cells with 1 mM N-ethylmaleimide without inhibiting the transport of AMP. Although no efflux was demonstrable in the absence of nucleotide in the medium, the intracellular adenine nucleotide pool could be exchanged with external unlabeled adenine nucleotides. Both ADP and ATP were as effective as AMP at inhibiting the uptake of [3H]AMP. Although this transport system was inhibited by low temperature (0 degrees C) and partially inhibited by the protonophore carbonyl cyanide-m-chlorophenyl hydrazone (1 mM), it was relatively insensitive to KCN (1 mM). The uptake of AMP at 34 degrees C had an apparent Kt for influx of 0.4 mM and a Vmax of 354 pmol min-1 per mg. At 0 degrees C there was a very rapid and unsaturable association of AMP with these organisms. Correction of the uptake data at 34 degrees C for the 0 degrees C component lowered the apparent Kt to 0.15 mM. Both magnesium and phosphate ions are required for optimal transport activity. Chemical measurements of the total intracellular nucleotide pools demonstrated that this system was not a net adenine nucleotide transport system, but that uptake of AMP was the result of an exchange with internal adenine nucleotides.     

Comparison of Methods for Extraction of Bacterial Adenine Nucleotides Determined by Firefly Assay

Adenine nucleotides in Escherichia coli, Bacillus cereus, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa were extracted using 10 different methods. Extracts were assayed for adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), and adenosine 5′-monophosphate (AMP) by the firefly method using an improved procedure. Analytical interference by bacterial enzymes not inactivated during the extraction was found to be a major problem. However, these enzymes were inactivated to a considerable extent by the inclusion of ethylenediaminetetraacetate in the extraction reagent. The 10 extraction methods were compared with respect to yield of adenine nucleotides, interference with the enzymic assay, reproducibility of the method, and stability of the extracts. Results indicated that extraction with trichloroacetic acid was the method most closely reflecting actual levels of ATP, ADP, and AMP in intact bacterial cells. However, for the extraction of ATP in some bacterial strains several other methods may be used and may be advantageous from a practical point of view.     

Human airway ecto-adenylate kinase. A mechanism to propagate ATP signaling on airway surfaces

Mechanically induced ATP release from human airway epithelial cells regulates mucociliary clearance through cell surface nucleotide receptors. Ectoenzymes detected on these cells were recently shown to terminate ATP-mediated responses by sequential dephosphorylation of extracellular ATP into ADP, AMP, and adenosine. We now demonstrate that an ecto-adenylate kinase (ecto-AK) contributes to the metabolism of adenine nucleotides on human airway epithelial surfaces by the reversible reaction: ATP + AMP 2ADP. This phosphotransferase exhibited a bilateral distribution on polarized primary cultures of human bronchial epithelial cells with a 4-fold higher activity on the mucosal surface. Ecto-AK presented an absolute requirement for magnesium and adenine-based nucleotides. UMP, GMP, and CMP could not substitute for AMP as gamma-phosphate acceptor, and UDP could not replace ADP. Apparent K(m) and V(max) values were 23 +/- 5 microM and 1.1 +/- 0.1 nmol x min(-1) x cm(-2) for ATP and 43 +/- 6 microM and 0.5 +/- 0.1 nmol x min(-1) x cm(-2) for ADP. Ecto-AK accounted for 20% of [gamma-(32)P]ATP dephosphorylation, and the impermeant AK inhibitor, diadenosine pentaphosphate, reduced ADPase activity by more than 70% on both epithelial surfaces. Time course experiments on ATP metabolism demonstrated that ecto-AK significantly prolongs effective ATP and ADP concentrations on airway epithelial surfaces for P2 receptor signaling and reduces by 6-fold adenosine production. Our data suggest a role for this nucleotide entrapment cycle in the propagation of purine-mediated mucociliary clearance on human airway epithelial surfaces.     

A novel mechanism for utilization of extracellular AMP in Vibrio parahaemolyticus

Vibrio parahaemolyticus could grow with AMP, ADP or ATP as the sole source of carbon. In the presence of Cl-, a membrane-bound Cl(-)-dependent 5'-nucleotidase seemed to hydrolyze the nucleotides extracellularly, and then the cells took up the resulting adenosine. In the absence of Cl-, although no significant dephosphorylation of the nucleotides occurred, the cells could still grow with AMP, but not with ADP or ATP. Moreover, in the presence of Cl-, Zn2+ inhibited the 5'-nucleotidase, and inhibited growth of the cells with ADP or ATP, but not with AMP, as the carbon source. V. parahaemolyticus was unable to grow with adenine or ribose 5-phosphate. These results suggested that the cells might have an AMP transport system. In fact, Na+ uptake was observed on addition of AMP to a cell suspension in the absence of Cl-, indicating Na+-AMP cotransport.     

Extracellular metabolism of nucleotides in neuroblastoma x glioma NG108-15 cells determined by capillary electrophoresis

1. The metabolism of extracellular nucleotides in NG108-15 cells, a neuroblastoma × glioma hybrid cell line, was studied by means of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC).2. In NG108-15 cells ATP, ADP, AMP, UTP, UDP, and UMP were hydrolyzed to the nucleosides adenosine and uridine indicating the presence of ecto-nucleotidases and ecto-phosphatases. The hydrolysis of the purine nucleotides ATP and ADP was significantly faster than the hydrolysis of the pyrimidine nucleotides UTP and UDP.3. ATP and UTP breakdown appeared to be mainly due to an ecto-nucleotide- diphosphohydrolase. ADP, but not UDP, was initially also phosphorylated to some extent to the corresponding triphosphate, indicating the presence of an adenylate kinase on NG108-15 cells. The alkaline phosphatase (ALP) inhibitor levamisole did not only inhibit the hydrolysis of AMP to adenosine and of UMP to uridine, but also the degradation of ADP and to a larger extent that of UDP. ATP and UTP degradation was only slightly inhibited by levamisole.4. These results underscore the important role of ecto-alkaline phosphatase in the metabolism of adenine as well as uracil nucleotides in NG108-15 cells. Dipyridamole, a potent inhibitor of nucleotide breakdown in superior cervical ganglion cells, had no effect on nucleotide degradation in NG108-15 cells.5. Dipyridamole, which is a therapeutically used nucleoside reuptake inhibitor in humans, reduced the extracellular adenosine accumulation possibly by allosteric enhancement of adenosine reuptake into the cells.     

Simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography with fluorescence derivatization

A sensitive method was developed for the simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography. The adenyl purines (adenine, adenosine, AMP, ADP, ATP and cyclic AMP) were derivatized using 2-chloroacetaldehyde for fluorescence detection, and the reaction and separation conditions were reinvestigated to improve sensitivity for small volume sample analysis. Each derivatized purine was separated on a Capcell Pack SG120A™ column with mobile phase consisting of 0.05 M citric acid–0.1 M dipotassium hydrogen phosphate (pH 4.0)–methanol (97+3). The detection limits were 100–1000 fmol/ml by fluorescence detection, some 500 times better than previous reports. The proposed method was applied to determine adenyl purines in human plasma. The purine levels were as follows: ATP (9.2–22.2 pmol/ml), ADP (5.5–22.2 pmol/ml), AMP (0.8–3.2 pmol/ml). Other purines, adenine, adenosine, cAMP were lower than 0.1 pmol/ml.     

Metabolism of adenosine, AMP and ATP in rats

Metabolism of [14C]adenosine in a dose of 100 mg per 1 kg of mass and [14C]ATP in the equimolar quantity was studied in rats after intraperitoneal administration. Adenosine is shown to enter tissues of the liver, spleen, thymus, heart and erythrocytes where it phosphorylates into adenine nucleotides (mainly ATP) and deaminates into inosine. The content of adenosine increases for a short period in the above tissues, except for erythrocytes and plasma. The latter accumulates a considerable amount of inosine and hypoxanthine, but only traces of uric acid, xanthine and adenine nucleotides. ATP administered to rats catabolizes through the adenosine formation. The exogenic adenosine and ATP replace in tissues and erythrocytes only a slight part (1-12%) of their total adenine nucleotide pool. The content of these metabolites and ADP in the blood plasma does not change essentially under the effect of adenosine, ATP and AMP. It is shown on rats whose adenine nucleotide pool of cells is marked by the previous administration of [14C]adenine that injections of adenosine, ATP and inosine do not accelerate catabolism of adenine nucleotides in tissues and erythrocytes as well as do not increase the level of catabolism products in the blood plasma. Adenosine enhances and ATP lowers the content of cAMP in spleen and myocardium, respectively.     

Uptake and Metabolism of [3H] Adenosine by Aplysia Ganglia and by Individual Neurons

[3H]Adenosine was taken up and metabolized by isolated ganglia of the marine mollusc Aplysia californica. After 2 h, most of the radioactivity was recovered as metabolites, including ATP, ADP, and AMP, as well as the deaminated products, inosine, hypoxanthine, and uric acid. Little remained in the form of adenosine. These pathways were not uniformly distributed among various tissue elements. In most individual neurons, inosine and its breakdown products were the principal metabolites of [3H]adenosine, whereas ATP and other nucleotides predominated in the connective tissue sheath. Endogenous levels of ATP, ADP, AMP, and adenosine in ganglia, sheath, and individual neurons were also determined using a fluorimetric-HPLC assay. The concentrations of the nucleotides were quite uniform in sheath and among the individual neurons assayed (1-5 pmol/microgram of protein); however, concentrations of adenosine were considerably higher in neurons than in the sheath.