Characterization of an ecto-5'-nucleotidase (EC 3.1.3.5) activity in intact cells of Trichomonas vaginalis

The enzymatic properties of an ecto-5'-nucleotidase were described in Trichomonas vaginalis. The enzyme hydrolyzes nucleoside monophosphates in optimum pH values of 7.5 and 6.5 for the 30236 strain and for the 30238 strain, respectively. Mg(2+) and Ca(2+) were activators of AMP hydrolysis in both strains. The apparent K(m) (Michaelis constant) values for Mg(2+)-AMP were 111.4+/-28.1 microM (mean+/-SD, n=3) for 30236 strain and 420.2+/-35.7 microM (mean+/-SD, n=3) for 30238 strain. The ecto-5'-nucleotidase activity was insensitive to levamisole and tetramisole, inhibitors of alkaline phosphatases, whereas alpha,beta-methylene-ADP inhibited the enzymatic activity of both strains. Our results showed that the AMP hydrolysis presents differences in some kinetic parameters between the two strains investigated. An analysis of the enzymatic chain involved in the ATP hydrolysis to adenosine will contribute to understanding the biochemical aspects of the parasite and the mechanisms related to host-parasite interactions.     

AMP hydrolysis in soluble and microsomal rat cardiac cell fractions: kinetic characterization and molecular identification of 5'-nucleotidase

The present study describes the enzymatic properties and molecular identification of 5'-nucleotidase in soluble and microsomal fractions from rat cardiac ventricles. Using AMP as a substrate, the results showed that the cation and the concentration required for maximal activity in the two fractions was magnesium at a final concentration of 1 mM. The pH optimum for both fractions was 9.5. The apparent K(m) (Michaelis constant) values calculated from the Eadie-Hofstee plot were 59.7+/-10.4 microM and 134.8+/-32.1 microM, with V(max) values of 6.7+/-0.4 and 143.8+/-23.8 nmol P(i)/min/mg of protein (means+/-S.D., n=4) from soluble and microsomal fractions respectively. Western blotting analysis of ecto-5'-nucleotidase revealed a 70 kDa protein in both fractions, with the major proportion present in the microsomal fraction. The presence of these enzymes in the heart probably has a physiological function in adenosine signalling. Furthermore, the presence of ecto-5'-nucleotidase in the microsomal fraction could have a role in the modulation of the excitation-contraction-coupling process through involvement of the Ca(2+) influx into the sarcoplasmic reticulum. The measurement of maximal enzyme activities in the two fractions highlights the potential capacity of the different pathways of purine metabolism in the heart.     

Reserpine attenuates interstitial adenosine-mediated activation of ecto-5'-nucleotidase in rat hearts in vivo

We examined whether reserpine-induced norepinephrine (NE) depletion attenuated the products of adenosine in rat heart. A flexibly mounted microdialysis technique was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase in rat hearts in situ. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and perfused with Tyrode solution containing adenosine 5'-monophosphate (AMP) at rate of 1.0 microliter/min. The baseline level of dialysate adenosine was 0.51 +/- 0.09 microM. The introduction of AMP (100 microM) through the probe increased markedly the dialysate adenosine to 8.95 +/- 0.86 microM, and this increase was inhibited by ecto-5'-nucleotidase inhibitor, alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP, 100 microM), to 0.66 +/- 0.38 microM. Thus, the level of dialysate adenosine is a measure of the ecto-5'-nucleotidase activity in the tissue in situ. AMP concentration for the half-maximal effect of adenosine release (EC(50)) was 107.3 microM. The maximum attainable concentration of dialysate adenosine (E(max)) by AMP was 21.1 microM. However, the EC(50) and E(max) values with reserpinized animals were 106.9 and 7.1 microM, respectively. Electrical stimulation of the left stellate ganglion increased significantly dialysate adenosine concentration, from the control level of 8.66 +/- 0.96 microM to 12.38 +/- 1.11 microM. After stimulation, dialysate adenosine returned to near the prestimulation level. When corresponding experiments were performed with reserpinized animals, the effect of electrical stimulation was abolished. Tyramine (endogenous catecholamine trigger) increased the adenosine concentration in a concentration-dependent manner. However, the elevation of adenosine concentration with reserpinized animals was not observed. These results suggest that reserpine attenuates NE-induced adenosine via stimulation of alpha(1)-adrenoceptor and protein kinase C mediated activation of ecto-5'-nucleotidase in rat heart.     

Role of adenosine deaminase, ecto-(5''-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.     

Immunologically distinct isoforms of ecto-5'-nucleotidase in nerve terminals of different areas of the rat hippocampus

Ecto-5'-nucleotidase is regarded as being the key enzyme in the formation of the neuromodulator adenosine from released ATP. However, the association of ecto-5'-nucleotidase with nerve terminals is not consensual. Only enzyme histochemical and biochemical studies, but not immunocytochemical studies, agree on a general synaptic location of the enzyme. To clarify this issue further we tested the effect of an antibody against ecto-5'-nucleotidase, previously used in immunocytochemical studies, on the activity of ecto-5'-nucleotidase in fractions of nerve terminals isolated from different areas of rat hippocampus. The specific activity of extracellular AMP catabolism was higher in synaptosomes from the CA3 area (0.81+/-0.06 nmol/min/mg of protein) than from synaptosomes from the CA1 area or the dentate gyrus or from the whole hippocampus (0.49-0.68 nmol/ min/mg of protein). The catabolism of AMP (10 microM) was equally inhibited (85-92%) in synaptosomes from whole hippocampus, CA1, CA3, or dentate gyrus by alpha,beta-methylene-ADP (100 microM) and equally unaffected by p-nitrophenyl phosphate (0.5 mM) or rabbit IgGs (100 microg/ml). However, the antiserum against ecto-5'-nucleotidase (100 microg/ml) inhibited extracellular AMP catabolism by 44% in CA3 synaptosomes but had little or no effect in synaptosomes from CA1, dentate gyrus, or whole hippocampus. A similar difference in the inhibitory potential of the antibody was observed between fractions of isolated 5'-nucleotidase binding to concanavalin A-Sepharose (70%) and fractions not retained by the lectin column (18%). Taken together, these results suggest that immunological isoforms of ecto-5'-nucleotidase exist in the rat hippocampal nerve terminals, with predominance in the CA3 area.     

Kinetic characterization of ATP diphosphohydrolase and 5'-nucleotidase activities in cells cultured from submandibular salivary glands of rats

The participation of ecto-ATP diphosphohydrolase (CD39; ecto-NTPDase) and ecto-5'-nucleotidase (CD73) activities in the nucleotide hydrolysis by salivary gland cells from rats was evaluated. We investigated the biochemical characteristics of these ectoenzymes in cells cultured from submandibular salivary glands of rats. The V(max) for the hydrolysis of ATP, ADP and AMP were 2275+/-153 (mean+/-SEM, n = 4), 941+/-96 (mean+/-SEM, n = 5) and 175+/-5 (mean+/-SEM, n = 5) nmol Pi liberated per min per mg of protein, respectively. The K(m) values for ATP, ADP and AMP were 224+/-8 microM (mean+/-SEM, n = 4), 163+/-15 microM (mean+/-SEM, n = 5) and 117+/-5 microM (mean+/-SEM, n = 5), respectively. The competition plot showed that ATP and ADP were hydrolyzed at the same active site on the enzyme. It may be postulated that the physiological role for this ecto-enzyme cascade is to terminate the action of the co-transmitter ATP, generating adenosine.     

Nucleotide metabolizing ecto-enzymes in Walker 256 tumor cells: molecular identification, kinetic characterization and biochemical properties

In this study we describe the molecular identification, kinetic characterization and biochemical properties of an E-NTPDase and an 5'-nucleotidase in Walker 256 cells. For the ATP, ADP and AMP hydrolysis there were optimum pH in the range 6.5-8.0, and absolute requirement for divalent cations (Mg(2+)>Ca(2+)). A significant inhibition of ATP and ADP hydrolysis was observed in the presence of high concentrations of sodium azide and 0.5 mM of Gadolinium chloride. These activities were insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors. The K(m) values were 464.2+/-86.6 microM (mean+/-SEM, n=4), 137.0+/-31 microM (mean+/-SEM, n=5) and 44.8+/-10.2 microM (mean+/-SEM, n=4), and V(max) values were 655.0+/-94.6 (mean+/-SEM, n=4), 236.3+/-27.2 (mean+/-SEM, n=5) and 177.6+/-13.8 (mean+/-SEM, n=5) nmol of inorganic phosphate min(-1) mg of protein(-1) for ATP, ADP and AMP, respectively. Using RT-PCR analysis we identified the mRNA of two members of the ecto-nucleoside triphosphate diphosphohydrolase family (NTPDase 2 and 5) and a 5'-nucleotidase. The presence of NTPDases and 5'-nucleotidase enzymes in Walker 256 tumor cells may be important to regulate the ratio adenine nucleotides/adenine nucleoside extracellularly, therefore motivating tumor growth.     

A novel transverse push-pull microprobe: in vitro characterization and in vivo demonstration of the enzymatic production of adenosine in the spinal cord dorsal horn

Adenosine produces analgesia in the spinal cord and can be formed extracellularly through enzymatic conversion of adenine nucleotides. A transverse push-pull microprobe was developed and characterized to sample extracellular adenosine concentrations of the dorsal horn of the rat spinal cord. Samples collected via this sampling technique reveal that AMP is converted to adenosine in the dorsal horn. This conversion is decreased by the ecto-5'-nucleotidase inhibitor, alpha,beta-methylene ADP. Related behavioral studies demonstrate that AMP administered directly to the spinal cord can reverse the secondary mechanical hyperalgesia characteristic of the intradermal capsaicin model of inflammatory pain. The specific adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT) inhibits the antihyperalgesia produced by AMP. This research introduces a novel microprobe that can be used as an adjunct sampling technique to microdialysis and push-pull cannulas. Furthermore, we conclude that AMP is converted to adenosine in the dorsal horn of the spinal cord by ecto-5'-nucleotidase and subsequently may be one source of adenosine, acting through adenosine A(1) receptors in the dorsal horn of the spinal cord, which produce antihyperalgesia.     

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.     

Presence of ectonucleotidases in cultured chromaffin cells: hydrolysis of extracellular adenine nucleotides

The granular ATP released from chromaffin cells during the secretory response can be hydrolyzed by ectonucleotidases that are present in the plasma membrane of these cells. The ecto-ATPase activity showed a Km for ATP of 250 +/- 18 microM and a VMAX value of 167 +/- 25 nmol/10(6) cells x min (1.67 mumol/mg protein x min) for cultured chromaffin cells, while the ecto-ADPase activity showed a Km value for ADP of 375 +/- 40 microM and a VMAX of 125 +/- 20 nmol/10(6) cells x min (1.25 mumol/mg protein x min). The ecto 5'-nucleotidase activity of cultured chromaffin cells was more specific for the purine nucleotides, AMP and IMP, than for the pirimidine nucleotides, CMP and TMP. The Km for AMP was 55 +/- 5 microM and the VMAX value was 4.3 +/- 0.8 nmol/10(6) cells x min (43 nmol/mg protein x min). The nonhydrolyzable analogs of ADP and ATP, alpha, beta-methylene-adenosine 5'-diphosphate and adenylyl-(beta, gamma-methylene)-diphosphonate were good inhibitors of ecto 5'-nucleotidase activity, the KI values being 73.3 +/- 3.5 nM and 193 +/- 29 nM, respectively. The phosphatidylinositol-specific phospholipase C released the ecto-5'-nucleotidase from the chromaffin cells in culture, thus suggesting an anchorage through phosphatidylinositol to plasma membranes. The presence of ectonucleotidases in chromaffin cells may permit the recycling of the extracellular ATP exocytotically released from these neural cells.     

Absolute rates of adenosine formation during ischaemia in rat and pigeon hearts.

1. The activities of ecto- and cytosolic 5'-nucleotidase (EC 3.1.3.5), adenosine kinase (EC 2.7.1.20), adenosine deaminase (EC 3.5.4.4) and AMP deaminase (EC 3.5.4.6) were compared in ventricular myocardium from man, rats, rabbits, guinea pigs, pigeons and turtles. The most striking variation was in the activity of the ecto-5'-nucleotidase, which was 20 times less active in rabbit heart and 300 times less active in pigeon heart than in rat heart. The cytochemical distribution of ecto-5'-nucleotidase was also highly variable between species. 2. Adenosine formation was quantified in pigeon and rat ventricular myocardium in the presence of inhibitors of adenosine kinase and adenosine deaminase. 3. Both adenosine formation rates and the proportion of ATP catabolized to adenosine were greatest during the first 2 min of total ischaemia at 37 degrees C. Adenosine formation rates were 410 +/- 40 nmol/min per g wet wt. in pigeon hearts and 470 +/- 60 nmol/min per g wet wt. in rat hearts. Formation of adenosine accounted for 46% of ATP plus ADP broken down in pigeon hearts and 88% in rat hearts. 4. The data show that, in both pigeon and rat hearts, adenosine is the major catabolite of ATP in the early stages of normothermic myocardial ischaemia. The activity of ecto-5'-nucleotidase in pigeon ventricle (16 +/- 4 nmol/min per g wet wt.) was insufficient to account for adenosine formation, indicating the existence of an alternative catabolic pathway.     

A specific inhibitor of heart cytosolic 5'-nucleotidase I attenuates hydrolysis of adenosine 5'-monophosphate in primary rat myocytes

ATP breakdown was triggered in primary rat myocytes in the presence of coformycin to force the catabolism of AMP through hydrolysis to adenosine. Selective inhibitors of the cytosolic 5'-nucleotidase I (c-N-I) from myocardium were used to measure the intracellular contribution of this enzyme to AMP hydrolysis under these conditions. The selective inhibitor 5-ethynyl-2',3'-dideoxyuridine inhibited the hydrolysis of AMP to adenosine in a concentration-dependent manner with an IC50 value of 20 microM. Maximal inhibition prevented 76% of the conversion of AMP to adenosine, indicating that under these conditions the majority of AMP hydrolysis in rat myocytes occurs through this enzyme. When ATP breakdown was triggered in the presence of thymidine 5'-phosphonate, a more potent inhibitor of the purified cytosolic 5'-nucleotidase, less inhibition of AMP hydrolysis occurred and only after prolonged preincubation of the myocytes with the inhibitor. These data demonstrate that the selective nucleoside inhibitors of c-N-I can effectively block the hydrolysis of AMP inside myocytes. Thus, these inhibitors may be useful tools in identifying the role of c-N-I during ATP catabolism in whole tissue and animal experiments.     

Ecto-5’-nucleotidase: Structure function relationships

Ecto-5'-nucleotidase (ecto-5'-NT) is attached via a GPI anchor to the extracellular membrane, where it hydrolyses AMP to adenosine and phosphate. Related 5'-nucleotidases exist in bacteria, where they are exported into the periplasmic space. X-ray structures of the 5'-nucleotidase from E. coli showed that the enzyme consists of two domains. The N-terminal domain coordinates two catalytic divalent metal ions, whereas the C-terminal domain provides the substrate specificity pocket for the nucleotides. Thus, the substrate binds at the interface of the two domains. Here, the currently available structural information on ecto-5'-NT is reviewed in relation to the catalytic properties and enzyme function.     

Effect of phosphate esters, nucleotides and nucleosides on 5'-nucleotidase of cultured mouse macrophages.

Mouse peritoneal macrophages elicited by intraperitoneal injection of sodium caseinate exhibit low levels of ecto-5'-nucleotidase (E. C. 3.1.3.5) activity in contrast to macrophages obtained by peritoneal lavage. When elicited cells were cultured under standard conditions in the presence of serum, a 2.5-fold increase in 5'-nucleotidase activity was observed over a period of 48 hours. Addition of adenosine monophosphate to the culture medium led to an augmented (5-fold) increase in the specific activity (per unit cell protein) as well as an absolute increase (per culture plate) of 5'-nucleotidase. Other adenosine-containing compounds also had stimulatory effects. The levels of this enzyme thus appear to be regulated by the extracellular levels of adenosine nucleotides. The product of the enzymatic reaction--adenosine--when added to the medium exhibited a toxic effect on these cells--as did adenosine monophosphate. However, the former substance did not augment the increase in enzyme activity during culture. The toxic effect could be suppressed when the cells were cultured in the presence of uridine 5'-monophosphate. The latter substance also depressed the stimulation of enzyme activity due to AMP.     

Effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis

Here we report the effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) hydrolysis was 5- to 7-fold higher for the fresh clinical strain, when compared with the ATCC (American Type Culture Collection) strain. ATP hydrolysis was activated in the presence of metronidazole in the ATCC strain, whilst it was inhibited 33% by 50 microM tinidazole in a fresh clinical isolate. The treatment of cells in the presence of metronidazole for 2 h inhibited ATP and ADP hydrolysis, whilst treatment with tinidazole inhibited ATP and ADP hydrolysis only in the fresh clinical isolate. The drugs did not change the ecto-5'-nucleotidase activity for both strains. Our results suggest that the modulation of extracellular ATP and ADP levels during treatment with these drugs could be a parasitic defence strategy as a survival mechanism in an adverse environment.     

Adenosine production and its interaction with protection of ischemic and reperfusion injury of the myocardium

Adenosine exerts cardioprotective effects on the ischemic myocardium. A flexibly mounted microdialysis probe was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase (a key enzyme responsible for adenosine production) in in vivo rat hearts. The level of adenosine during perfusion of adenosine 5'-adenosine monophosphate (AMP) was given as an index of the activity of ecto-5'-nucleotidase in the tissue. Endogenous norepinephrine (NE) activates both alpha(1)-adrenoceptors and protein kinase C (PKC), which, in turn, activates ecto-5'-nucleotidase via phosphorylation thereby enhancing the production of interstitial adenosine. Histamine-release NE activates PKC, which increased ecto-5'-nucleotidase activity and augmented release of adenosine. Opening of cardiac ATP sensitive K(+) (K(ATP)) channels may cause hydroxyl radical (.OH) generation through NE release. Lysophosphatidylcholine (LPC), an endogenous amphiphiphilic lipid metabolite, also increases the concentration of interstitial adenosine in rat hearts, through the PKC-mediated activation of endogenous ecto-5'-nucleotidase. Nitric oxide (NO) facilitates the production of interstitial adenosine, via guanosine 3',5'-cyclic monophosphate (cGMP)-mediated activation of ecto-5'-nucleotidase as another pathway. These mechanisms play an important role in high sensitivity of the cardiac adenosine system. Adenosine plays an important role as a modulator of ischemic reperfusion injury, and that the production and mechanism of action of adenosine are linked with NE release.     

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.     

NTPDase and 5' ecto-nucleotidase expression profiles and the pattern of extracellular ATP metabolism in the Walker 256 tumor

In this study, we evaluated the NTPDases and ecto-5'-nucleotidase (CD73) expression profiles and the pattern of adenine nucleotide hydrolysis in rats submitted to the Walker 256 tumor model, 6, 10 and 15 days after the subcutaneous inoculation. Using RT-PCR analysis, we identified mRNA for all of the members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated and a 5'-nucleotidase. By quantitative real-time PCR, Entpd1 (Cd39) and Entpd2 (Cd39L1) and CD73 were identified as the dominant genes expressed by the Walker 256 tumor, at all times studied. Extracellular adenine nucleotide hydrolysis by the Walker 256 tumor was estimated by HPLC analysis. Rapid hydrolysis of extracellular ATP by the tumor cells was observed, leading to the formation of adenosine and inosine in cells obtained from solid tumors at 6 and 10 days after inoculation. Cells obtained from solid tumors at 15 days of growth presented high levels of AMP and presented adenosine as a final product after 90 min of incubation. Results demonstrate that the presence of NTPDases and 5'-nucleotidase enzymes in Walker 256 tumor cells may be important for regulation of the extracellular adenine nucleotides/adenine nucleoside ratio, therefore leading to tumor growth.     

Ecto-5''-Nucleotidase Is Associated with Cholinergic Nerve Terminals in the Hippocampus but Not in the Cerebral Cortex of the Rat

The extracellular catabolism of exogenously added AMP was studied in immunopurified cholinergic nerve terminals and in slices of the hippocampus and cerebral cortex of the rat. AMP (10 microM) was catabolized into adenosine and inosine in hippocampal cholinergic nerve terminals and in hippocampal slices, as well as in cortical slices. IMP formation from extracellular AMP was not detected. alpha, beta-Methylene ADP (100 microM) inhibited almost completely the extracellular catabolism of AMP in these preparations. The relative rate of catabolism of AMP was greater in hippocampal slices than in cortical slices. AMP was virtually not catabolized when added to immunopurified cortical cholinergic nerve terminals, although ATP could be catabolized extracellularly under identical conditions. The comparison of the relative rates of catabolism of exogenously added AMP, calculated from the amount of AMP catabolized after 5 min, in hippocampal cholinergic nerve terminals and in hippocampal slices revealed a nearly 50-fold enrichment in the specific activity of ecto-5'-nucleotidase upon immunopurification of the cholinergic nerve terminals from the hippocampus. The results suggest that there is a regional variation in the subcellular distribution of ecto-5'-nucleotidase activity in the rat brain, the ecto-5'-nucleotidase in the hippocampus being closely associated with the cholinergic nerve terminals, whereas in the cerebral cortex ecto-5'-nucleotidase activity seems to be located preferentially outside the cholinergic nerve terminals.