Stimulus- and cell-type-specific release of purines in cultured rat forebrain astrocytes and neurons

Adenosine is formed during conditions that deplete ATP, such as ischemia. Adenosine deaminase converts adenosine into inosine, and both adenosine and inosine can be beneficial for postischemic recovery. This study investigated adenosine and inosine release from astrocytes and neurons during chemical hypoxia or oxygen-glucose deprivation. In both cell types, 2-deoxyglucose was the most effective stimulus for depleting cellular ATP and for evoking inosine release; in contrast, oxygen-glucose deprivation evoked the greatest adenosine release. alpha,beta-Methylene ADP, an inhibitor of ecto-5'nucleotidase, significantly reduced adenosine release from astrocytes but not neurons. Dipyridamole, an inhibitor of equilibrative nucleoside transporters, inhibited both adenosine and inosine release from neurons. Erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase, reduced neuronal inosine release evoked by oxygen-glucose deprivation but not by 2-deoxyglucose treatment. These data indicate that (1). astrocytes release adenine nucleotides that are hydrolyzed extracellularly to adenosine, whereas neurons release adenosine per se, (2). inosine is formed intracellularly and released via nucleoside transporters, and (3). inosine is formed by an adenosine deaminase-dependent pathway during oxygen-glucose deprivation but not during 2-deoxyglucose treatment. In summary, the metabolic pathways for adenosine formation and release were cell-type dependent whereas the pathways for inosine formation were stimulus dependent.     

Cytomegalovirus infection increases the expression and activity of ecto-ATPase (CD39) and ecto-5'nucleotidase (CD73) on endothelial cells

We describe enhanced expression and enzymatic activity of ecto-ATPase and ecto-5'nucleotidase on CMV infected endothelial cells as compared to uninfected cells. These ectoenzymes play a major role in modulation of platelet activation and aggregation. Furthermore, adenosine has a modulatory effect upon inflammation. Addition of ATP, ADP or AMP to cultures of CMV infected or uninfected endothelial cells revealed increased turnover of AMP in CMV infected endothelial cells. In addition, the superoxide production by stimulated polymorphonuclear cells was inhibited in the presence of CMV infected endothelial cells as compared to uninfected cells, probably due to the enhanced activity of ecto-5'nucleotidase and associated to production of adenosine.     

A kinetic study of the rat liver adenosine kinase reverse reaction

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP --> AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP --> ATP + inosine + NH(3). The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.     

A Kinetic Study of the Rat Liver Adenosine Kinase Reverse Reaction

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH₃. The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.     

Stimulation of maximal intracellular insulin action on glycogen synthase by preincubation of adipocytes with adenosine 5'-triphosphate

Preincubation of rat adipocytes with ATP further stimulated maximal insulin action on glycogen synthase. Half-maximum concentration of ATP was 5 X 10(-5) M. ATP, ADP, adenosine, inosine, and GTP were effective, while beta-gamma-methylene ATP was without effect. ADP and GTP were less potent than ATP, adenosine, or inosine. Inosine was active without insulin but was without effect in the presence of insulin. The mechanism of action of adenosine was clearly different from ATP. While ATP required both Mg2+ and Ca2+ for effectiveness, adenosine required only Ca2+. The effect of ATP, but not of adenosine, was preserved after cells were washed. The adenosine effect was completely blocked by theophylline, but the ATP effect was inhibited only 40%. The ATP effect was thus not due to adenosine generated by ATP breakdown.     

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.     

Effect of age and day time on the adenosine modulation of basal and insulin-stimulated glucose transport in rat adipocytes

1. The relationship between the activity of adenosine metabolizing enzymes 5'nucleotidase (5'N), adenosine kinase (A.K.) and adenosine deaminase (A.D.) with basal and insulin-stimulated glucose transport in isolated fat cells from young and old animals was studied at 08:00 and 16:00 hr. 2. In cells from young animals a larger insulin-stimulation of glucose transport was observed at 16:00 hr than at 08:00 hr. Also at 16:00 hr small changes in 5'N, A.K. and A.D. activities suggest a decrease in adenosine formation. 3. In the cells from old animals no effect of insulin was observed at any time, while a 3-5-fold increase in 5'N indicated a predominance of adenosine formation at both times studied. 4. An inverse relationship was observed in the changes of adenosine metabolism and insulin action.     

Flow system for fish freshness determination based on double multi-enzyme reactor electrodes

A double reactor system for the determination of fish and shellfish freshness using the freshness indicator, K-value (K=[(HxR+Hx)/(ATP+ADP+AMP+IMP+HxR+Hx)]x100), was developed, where ATP, ADP, AMP, IMP, HxR and Hx are adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosine monophosphate, inosine and hypoxanthine, respectively. The system consisted of a pair of enzyme reactors with an oxygen electrode positioned close to the respective reactor. The enzyme reactor (I) was packed with nucleoside phosphorylase and xanthine oxidase immobilized simultaneously on chitosan beads (immobilized enzyme A). Similarly, the enzyme reactor (II) was packed with immobilized enzyme A and immobilized enzyme B (co-immobilized alkaline phosphatase and adenosine deaminase). Moreover, this reactor consisted of two layers, the enzyme A and enzyme B (1:1). A good correlation was obtained between K values, which were determination by the proposed system and by the HPLC method. One assay could be completed within 5 min. The signal for the determination of K value of fish and shellfish was reproducible within 2.3%. The long-term stability of the enzyme reactors was evaluated at 30 degrees C for 28 days.     

Studies on 5′-Nucleotidase-Lacking Mutants Derived from Bacillus subtilis

Two 5′-nucleotidase-lacking mutants, R–42 and A–1, were derived from an adenine-requiring mutant, B. subtilis 1145–2–83, which has productivity of both inosine and hypoxanthine. Strain A–1 accumulated 5′-IMP as well as inosine and hypoxanthine, and strain R–42 accumulated 5′-IMP and 5′-GMP as well as inosine and hypoxanthine in their culture fluids. These mutants responded to either adenine or adenosine, but did not to 5′-AMP. This fact suggests that adenine or adenosine may be incorporated into the cells, but 5′-AMP may neither be incorporated into the cells nor be degraded during culture. 5′-GMP was converted to 5′-IMP, and 5′-AMP was phosphrylated to ADP in the growing culture of strain A–1.     

Detection and determination of adenosine diphosphate and related substances in plasma

1. A method is described for detecting and determining the products of metabolism of ADP added to plasma at initial concentrations of about 1mum-ADP. 2. ATP, ADP, AMP, adenosine, inosine and hypoxanthine were detected in human platelet-rich plasma after incubation with ADP and in the presence of either heparin or heparin-citrate. 3. The products of incubation of ADP with human platelet-poor plasma in the presence of heparin were the same as with platelet-rich plasma, except that, when the initial concentration of ADP was 1.5mum, little or no ATP was detected. 4. The ATP detected in platelet-rich plasma when 1.5mum-ADP was initially incubated was present in the platelets and not in the plasma. 5. The time for 50% decay of ADP in either platelet-rich or platelet-poor plasma in the presence of heparin was about 20min. when the initial concentration of ADP was 200mum, but was 6-9min. when the initial ADP concentration was 1.5-2.5mum. The corresponding values in the presence of heparin-citrate were about 45min. and about 9-12min. respectively. 6. Hypoxanthine accumulated to a greater extent in platelet-rich than in platelet-poor plasma after the addition of ADP. 7. After incubation for 15-20min. of either platelet-rich plasma or suspensions of washed platelets in saline with adenosine at an initial concentration of about 3-4mum, ATP, ADP and AMP were detected in the platelets. Similar incubations of washed platelets with inosine also showed the formation of these substances, but to a much less extent. 8. After the addition of adenosine to suspensions of washed platelets in saline, inosine and hypoxanthine were detected in the incubation mixture. After the addition of inosine, hypoxanthine was detected. 9. When ADP at an initial concentration of 1.5mum was added to platelet-rich plasma containing adenosine deaminase, no adenosine was detected in the incubation mixture. There was no difference in the rate of decay of ADP in the presence or absence of the deaminase, but ATP formation was decreased in its presence.     

Immunological evidence that plasma-membrane 5'-nucleotidase is a transmembrane protein

Antibodies inhibiting specifically plasma membrane-bound 5'nucleotidase were used to determine the disposition of the enzyme in various lymphoma cell lines. Fluorescent Fab fragments of the inhibiting antibodies bound to MF2s and MOPC 173 plasmacytoma cells, whereas no fluorescence was observed with P 1798 thymoma cells in which the enzyme was absent. The relative potency of the antiserum in inhibiting the enzyme in right-side-out and inside-out plasma membrane vesicles prepared from the above cell lines indicated that various group of antigenic determinants were present. One group of antigenic determinants was present at the external face of the cell, and a second was associated with the inner surface of the membrane. A third group of antigenic determinants was located in the vicinity of the active site of the enzyme and it is the group that varied in the various plasmacytoma cells studied. The results are interpreted as immunological evidence that 5'nucleotidase is a transmembrane glycoprotein.     

Inhibition of lipolysis and cyclic AMP accumulation by adenosine analogues in hamster epididymal adipocytes exposed to cholera toxin

The effects of adenosine, N6-phenylisopropyl adenosine and 2',5'-dideoxyadenosine on lipolysis and cyclic AMP accumulation, in hamster adipocytes treated with cholera toxin, were studied. Cholera toxin caused an increase in lipolysis and cyclic AMP accumulation that was dependent upon the concentration of toxin and the length of time cells were exposed to the toxin. When N6-phenylisopropyl adenosine or 2',5'-dideoxyadenosine were present, the lipolytic and cyclic AMP responses to cholera toxin were inhibited. The adenosine analogues were equally effective inhibitors of lipolysis and cyclic AMP accumulation, when they were added 1 or 2 h after exposure to the toxin. Enzymatic removal of endogenously produced adenosine with adenosine deaminase potentiated both the lipolytic and cyclic AMP responses to cholera toxin. In addition, the inhibitory effects of N6-phenylisopropyl adenosine, 2'5'-dideoxyadenosine and clonidine on lipolysis and cyclic AMP were enhanced consequent to enzymatic removal of adenosine. These data show responses of intact fat cells to N6-phenylisopropyl adenosine, 2',5'-dideoxyadenosine or removal of endogenous adenosine and provide evidence for an adenosine sensitivity of fat cells exposed to cholera toxin.     

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.     

Compartmentalized ATP pools produced from adenosine are nuclear pools

Incubation of African green monkey kidney (BS-C-1) cells and mouse fibroblasts (3T6) in the presence of adenosine for 4 hours resulted in increases in the nuclear compartment pools of adenosine 5'-triphosphate (ATP) and nuclear ATP/adenosine 5'-diphosphate (ADP) ratios. Adenine and inosine, which yield increases in total cellular ATP pools and ATP/ADP ratios similar to those promoted by adenosine, do not produce similar increases in the nuclear compartment. Adenosine-promoted increases in nuclear ATP pools were higher in the untransformed, serially propagated, BS-C-1 cells than in the spontaneously transformed 3T6 cells. Adenosine-promoted compartmentalized ATP pools in primary chick embryo fibroblasts were reduced upon transformation of these cells with Rous sarcoma virus, resulting in free mixing of all of the ATP pools synthesized from various salvage precursors. The growth regulatory properties of the nuclear compartment pools of adenine nucleotides is suggested by the big increases in nuclear ATPase and adenosine 5'-monophosphate (AMP) deaminase activities upon the entry of 3T6 cells into the S phase of their cycle. These enzymatic activities would tend to lower the nuclear ATP/ADP ratios and reduce the total adenine nucleotide pools in these nuclei respectively--conditions which were shown by earlier in vitro studies to be favorable to DNA replication.     

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.     

Phosphorylation of cytokinin by adenosine kinase from wheat germ

Adenosine kinase was partially purified from wheat germ. This enzyme preparation, which was devoid of adenine phosphoribosyltransferase and nearly free of adenosine deaminase but contained adenylate kinase, rapidly phosphorylated adenosine and a cytokinin, N⁶-(δ²-isopentenyl)adenosine. Electrophoretic analysis indicated that only N⁶-(δ²-isopentenyl)adenosine-monophosphate was formed from the cytokinin while about 55% AMP, 45% ADP, and a trace of ATP were formed from adenosine. The biosynthesized nucleoside monophosphates were quantitatively hydrolyzed to the corresponding nucleosides by 5′-nucleotidase and the isopentenyl side chain of the phosphorylated cytokinin was not cleaved. The enzyme did not catalyze phosphorylation of inosine.     

Biological variation of rabbit chondrocyte 5'nucleotidase

The activity of 5'nucleotidase was investigated in rabbit chondrocytes. Studies of chondrocytes in organ culture and intact cells revealed that ecto-5'nucleotidase activity varies with the age of the animal. Specific activities of 0.29, 0.62, 1.97, and 1.39 mumole/hr/10(6) cells were observed in intact cells from 3-week-, 3-month-, 6-month-, and 2-year-old animals. Specific activities of 15.5, 33.1, 80.3, and 39.5 mumole/hr/mg protein, respectively, were observed when the enzyme protein was solubilized from disrupted cells with the detergent octyl-beta-D-glucopyranoside. Membrane-bound nonspecific neutral phosphatase activity did not vary with age. Activity of 5'nucleotidase was observed on all chondrocytes using a histochemical staining technique. Kinetic properties of the enzyme from animals of different ages were investigated. No differences were noted in apparent Km value for AMP (23 microM), pH optima (7.3), substrate competition studies, and effects of MgCl2 or CaCl2. These data demonstrate a biological variation in rabbit chondrocyte 5'nucleotidase activity with age. The variation of activity represents an apparent increase in the amount of enzyme protein rather than an alteration in kinetic properties.     

Biosynthesis of bacterial glycogen: activator specificity of the adenosine diphosphate glucose pyrophosphorylases from the genus Rhodospirillum.

The adenosine diphosphate (ADP) glucose pyrophosphorylases from Rhodospirillum fulvum, Rhodospirillum molischianum, and Rhodospirillum tenue were partially purified, and their kinetic properties were studied. The enzyme from the three organisms was found to be activated by pyruvate and thus was similar to the Rhodospirillum rubrum enzyme that had been previously studied (C. E. Furlong, and J. Preiss, J. Biol. Chem. 244:2539-2548, 1979). The enzymes from R. fulvum, R. molischianum, and R. tenue were also activated by oxamate, an analog of pyruvate. Other alpha-keto acids, alpha-ketobutyrate and hydroxypyruvate, activated to a smaller extent. The presence of pyruvate increased the apparent affinity for adenosine 5'-triphosphate and MgCl2 for all three enzymes. The R. molischianum enzyme has very little sensitivity to inhibition by adenosine 5'-monophosphate, ADP, or inorganic phosphate. However, R. tenue ADPglucose pyrophosphorylase is very sensitive to inhibition by adenosine 5'-monophosphate, and the R. fulvum enzyme is inhibited by ADP. Increasing pyruvate concentration reversed the inhibition caused by adenosine 5'-monophosphate or ADP. Since ADPglucose is the glycosyl donor for synthesis of glycogen, it is possible that in vivo glycogen synthesis is regulated by the concentration of pyruvate and, in the case of R. fulvum and R. tenue, by the ratio of pyruvate concentration to inhibitor concentration.     

Purine accumulation in human fat cell suspensions. Evidence that human adipocytes release inosine and hypoxanthine rather than adenosine

Human adipocytes are of limited viability (7 +/- 2% release of lactate dehydrogenase/h) and contain active ectophosphatases which are capable of sequentially degrading ATP to adenosine. At densities of 30,000-40,000 cells/ml, human fat cell suspensions accumulated adenosine, inosine, and hypoxanthine, and their concentrations were 38 +/- 8, 120 +/- 10, and 31 +/- 7 nmol/liter after 3 h of incubation. Dipyridamole (10 mumol/liter), an inhibitor of nucleoside transport, caused a 5-7-fold increase in adenosine accumulation which was reduced by 85% on inhibition of ectophosphatases by beta-glycerophosphate and antibodies against ecto-5'-nucleotidase or alpha, beta-methylene 5'-adenosine diphosphate (10 mumol/liter), respectively, indicating that most of the adenosine is produced in the extracellular compartment. Accordingly, the spontaneous accumulation of adenosine was reduced beyond 5 nmol/liter on inhibition of ectophosphatase activities or removal of extracellular AMP by AMP deaminase (4 units/ml). Added adenosine (30 nmol/liter) disappeared until its concentration approached 5 nmol/liter. Isoproterenol (1 mumol/liter) had no effect on adenosine accumulation regardless whether purine production from extracellular sources was minimized or not. In contrast to adenosine, the concentrations of inosine and hypoxanthine displayed only a modest decrease (30-50%) on inhibition of ectophosphatase activities. In addition, isoproterenol caused a 2-3-fold increase in inosine and hypoxanthine production which was concentration-dependent and could be inhibited by propranolol. It is concluded that the adenosine that accumulates in human adipocyte suspensions is almost exclusively derived from adenine nucleotides which are released by leaking cells. By contrast, inosine and hypoxanthine are produced inside the cells, and the release of these latter purines appears to be linked to ATP turnover via adenylate cyclase.     

Nucleoside transport and metabolism in erythrocytes from the Yucatan miniature pig. Evidence that inosine functions as an in vivo energy substrate

Erythrocytes from the Yucatan miniature pig, like those from the normal domestic pig, lack functional glucose transporters and were unable to utilize plasma glucose as an energy source. In contrast, inosine and adenosine entered the cells rapidly. The nucleoside transporter responsible for this uptake was identified as a band 4.5 polypeptide (5000 copies per cell; apparent Mr 45 000-66 000). Inosine concentrations in the physiological plasma range (1.6-2.5 microM) were found to maintain normal erythrocyte ATP levels and ATP/ADP ratios during prolonged in vitro incubation of cells at 37 degrees C, an effect that was blocked by the specific nucleoside transport inhibitor, nitrobenzylthioguanosine. In the absence of extracellular nucleoside, cells 'protected' themselves against some of the consequences of deprivation of energy substrate by glycolyzing the ribose moiety of inosine produced during ATP catabolism. Although erythrocytes from the miniature pig were capable of utilizing extracellular adenosine as an energy substrate, plasma samples from these animals contained less than 0.4 microM adenosine. It is concluded that inosine is a major physiological energy source of pig erythrocytes.