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PubMed1992

Prenatal exposure to ethanol alters plasma membrane glycoproteins of astrocytes during development in primary culture as revealed by concanavalin A binding and 5'-nucleotidase activity.

We have investigated the effect of prenatal exposure to ethanol on the extent of binding and surface distribution of the lectin concanavalin A (con A) on rat cortical astrocytes during the periods of proliferation and differentiation in primary culture. The enzymatic activity of the plasma membrane glycoprotein 5'-nucleotidase was also assessed. The cells were obtained from control fetuses (no exposure to ethanol) and from fetuses prenatally exposed to ethanol. The main findings were: 1) both proliferating and differentiating control astrocytes showed two distinct types of surface con A receptors that could correspond to high- and low-affinity binding sites; 2) the extent of con A binding was greater in mature than in proliferating control cells; 3) the distribution of con A on cell surface components changed with differentiation; 4) the activity of 5'-nucleotidase showed a substantial increment during the period of differentiation; and 5) prenatal exposure to ethanol clearly decreased the ability of astrocytes to bind con A, altered the surface distribution of the receptors for this lectin, and decreased the activity of 5'-nucleotidase. These effects were more marked in proliferating cells. In conclusion, it is shown that the extent of con A labeling and the activity of 5'-nucleotidase in astrocytes are dependent on the stage of cell differentiation and that prenatal exposure to ethanol alters the plasma membrane structure of these cells during development.

5'-Nucleotidase↗
PubMed1980

Studies on AMP deaminase and 5'-nucleotidase in rat brain under different experimental conditions.

Adenosine monophosphate (AMP) deaminase and 5'-nucleotidase, the two enzymes involved in the disposal of AMP, have been detected in different regions of normal rat brain and in animals subjected to heightened neuronal activity (leptazol-induced convulsions) and to depression of the central nervous system (CNS) by the administration of barbiturates. They have also been estimated in the CNS of animals subjected to anoxia or treated with lithium and ammonium salts. The AMP deaminase activity was found to be highest in cerebellum and lowest in cerebral cortex, while the 5'-nucleotidase activity was found to be highest in brain stem and lowest in cerebellum. The AMP deaminase activity was elevated in all the regions of brain during the preconvulsive and convulsive periods. The activity returned to normal during recovery. The activity of 5'-nucleotidase was found to be depressed in the preconvulsive and post-convulsive periods. The enzyme was also found to be depressed in all the three regions after the administration of barbiturates. Administration of lithium or ammonium salts of induction of anoxic states resulted in an increase in the activity of AMP deaminase in all the three regions of brain. These results are discussed in relation to the probable production of cyclic AMP and cyclic guanosine monophosphate (GMP) which may have depressive and excitatory roles, respectively, in brain. It appears that increased AMP deaminase activity is associated with increased neuronal activity while depression of 5'-nucleotidase activity is associated with conditions of decreased CNS excitability.

AMP Deaminase↗
PubMed1993

8-Azido-adenine nucleotides as substrates of ecto-nucleotidases in chromaffin cells: inhibitory effect of photoactivation.

The components of the ecto-nucleotidase pathway at the extracellular surface of adrenal chromaffin cells are the enzymatic activities responsible for the hydrolysis of granular nucleotide compounds released during the secretory response. The azido-nucleotides have been largely employed to characterize nucleotide binding sites. The 8-azido-adenine nucleotides were studied as substrates of ecto-nucleotidases in cultured chromaffin cells by HPLC procedures. 8-Azido-ATP (8-N3-ATP) was a good substrate for ecto-ATPase activity, the Km value was 256.30 +/- 36.41 microM, and the Vmax value was 14.33 +/- 0.84 nmol/min x 10(6) cells. 8-Azido-ADP (8-N3-ADP) was dephosphorylated by the ecto-ADPase activity with a Km value of 595.29 +/- 67.44 microM and Vmax value of 6.86 +/- 0.45 nmol/min x 10(6) cells. These kinetic parameters were similar to those obtained with ATP and ADP in the same culture and incubation conditions. 8-Azido-AMP (8-N3-AMP) was not hydrolyzed by the ecto-5'-nucleotidase activity. The 8-azido-nucleotides competitively inhibited the hydrolysis of adenine nucleotides, with Ki values in the same range as the Km. After uv photoactivation, the three 8-azido-nucleotides (100 microM) irreversibly inhibited and to a similar extent, between 40 and 55%, each of ecto-nucleotidase activities. UV photoactivation in the presence of nucleotides in the same concentration range was an effective protection from the inhibition.

5'-Nucleotidase↗
PubMed2000

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.

5'-Nucleotidase↗
PubMed1999

Induction of human high K(M) 5'-nucleotidase in cultured 293 cells.

Human 293 cells were stably transfected with a plasmid introducing a receptor for the ecdysone analog muristerone. The cells were further stably transfected with muristerone-inducible expression vectors carrying either the cDNA for the human high K(M) 5'-nucleotidase or the coding sequence of the nucleotidase linked to the 5'-end of the sequence for the green fluorescent protein. Upon induction, both types of transfectants overproduced nucleotidase activity in a time- and dose-dependent manner. Western blots gave values close to the expected subunit molecular masses of 65 and 92 kDa, respectively, excluding processing of the induced proteins. Cells induced to overexpress the nucleotidase showed a decreased growth rate and contained smaller pools of each of the four common ribonucleoside triphosphates. They showed no increased resistance to the toxicity of 2-chlorodeoxyadenosine.

5'-Nucleotidase↗
PubMed1989

Distribution of 5'-nucleotidase in the renal interstitium of the rat.

The hydrolysis of 5'-AMP by 5'-nucleotidase is the main source of adenosine. In various tissues adenosine is a local mediator adjusting the organ work to the available energy. In the kidney it regulates renal hemodynamics, glomerular filtration rate and renin release via specific receptors of the arteriolar walls. By immunocytochemistry we identified interstitial and tubular sites of 5'-nucleotidase in the rat kidney. In the interstitium the enzyme was detected only in the cortical labyrinth, the compartment that comprises all arteriolar vessels besides other putative targets of adenosine. The 5'-nucleotidase-positive cells of the interstitium were identified as fibroblasts. The fibroblasts are in close contact with the tubules as well as with the vessels. Thus, any 5'-AMP released by the tubules into the interstitial space would be converted to adenosine in the direct vicinity of its assumed targets. Adenosine produced by tubular cells would hardly have access to its known targets, since 5'-nucleotidase is restricted to the luminal cell surface. Pathological events affecting the fibroblasts might influence renal function by modifying the interstitial adenosine production.

5'-Nucleotidase↗
PubMed1994

Reconstitution of 5'-nucleotidase of bull seminal plasma in spin-labeled liposomes.

Seminal plasma separated from freshly ejaculated bull semen contains vesicles with a 5'-nucleotidase activity incorporated as an ectoenzyme anchored by glycosyl phosphatidylinositol (GPI). After its extraction from bull seminal plasma vesicles, the protein was purified and reconstituted into hen egg yolk lecithin liposomes obtained through prolonged dialysis of buffered n-octylglucoside detergent solutions of lipid, protein and various effectors against detergent-free solutions. Gel filtration experiments showed that the enzyme incorporated into liposomes in a dimeric form with its two subunits linked by disulfide bridges. In the presence of reduced glutathione, the protein dissociated into monomers and failed to incorporate into liposomes. Electron spin resonance (ESR) experiments, performed with liposomes containing electron spin labels localized at the hydrophilic lipid headgroups (5-doxyl stearic acid) or in the hydrophobic lipid hydrocarbon chains (16-doxyl stearic acid), demonstrated that the incorporation of 5'-nucleotidase resulted in the immobilization of the spin probes. Furthermore, the spectral parameters obtained before and after treatment of 5'-nucleotidase-containing liposomes with phosphatidylinositol-specific phospholipase C (PI-PLC) indicated that the liposome membrane bilayer did not contain protein segments. This supports the well-known ecto-localization of 5'-nucleotidase and rules out a previously reported possibility of a proteic transmembrane anchoring of the enzyme.

5'-Nucleotidase↗
PubMed1976

Hereditary hemolytic anemia with erythrocyte pyrimidine 5'-nucleotidase deficiency in Spain. Clinical, biological and familial studies.

We report a hereditary hemolytic anemia associated with a severe erythrocyte pyrimidine 5'-nucleotidase deficiency in a Spanish family of five members in which the parents are first cousins. Both parents exhibited decreased nucleotidase activity without clinical or hematologic abnormalities. Two children (a male and a female) showed severe pyrimidine 5'-nucleotidase deficiency with hemolytic anemia. The remaining child (a male) showed no signs of the disease. The findings strongly suggest an autosomal recessive mode of inheritance in this enzymopathy. This seems to be the first report of pyrimidine 5'-nucleotidase deficiency in Spain.

Adenosine Triphosphate↗
PubMed1979

Histochemistry in psoriasis. 5'-Nucleotidase in psoriatic parakeratotic horny layer.

The 5'-nucleotidase activity in psoriatic and normal human epidermis was studied in comparison to acid phosphatase activity. The optimum pH in normal human epidermis was about 5.0 at room temperature. The activity of both enzymes was found to be high in the transitional zone. Acid phosphatase (non-specific) activity was strongly positive in the psoriatic parakeratotic horny layers whereas 5'-nucleotidase activity in that area was completely absent. The results suggest that the enzyme which degrades nucleoside-5'-phosphate to nucleoside and inorganic phosphate is not acid phosphatase but 5'-nucleotidase. Nuclear preservation in psoriatic hyperkeratosis was attributed to absence or inactivation of specific enzymes of nuclear degradation, such as 5'-nucleotidase, rather than acid phosphatase.

Acid Phosphatase↗
PubMed1990

Lipid peroxidation as the mechanism of modification of brain 5'-nucleotidase activity in vitro.

The effect of lipid peroxidation on the Mg2(+)-independent and Mg2(+)-dependent activity of brain cell membrane 5'-nucleotidase was determined and the affinity of the active sites of Mg2(+)-dependent enzyme for 5'-AMP (substrate) and Mg2+ (activator) was examined. Brain cell membranes were peroxidized at 37 degrees C in the presence of 100 microM ascorbate and 25 microM FeCl2 (resultant) for 10 min. The activity of 5'-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20 +/- 0.10 to 17.5 +/- 1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg2(+)-independent 5'-nucleotidase increased from 0.201 +/- 0.020 in controls to 0.305 +/- 0.028 mumol Pi/mg protein/hr in peroxidized membranes. In the presence of 10 mM Mg2+, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control. In peroxidized preparation, the affinity of active site of Mg2(+)-dependent 5'-nucleotidase for 5'-AMP tripled, as indicated by a significant decrease in Km (Km = 95 +/- 2 microM AMP for control; Km = 32 +/- 2 microM AMP for peroxidized). Vmax was significantly reduced from 3.35 +/- 0.16 in control to 1.70 +/- 0.9 mumoles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg2+ significantly increased (Km = 6.17 +/- 0.37 mM Mg2+ for control; Km = 4.0 +/- 0.31 peroxidized).(ABSTRACT TRUNCATED AT 250 WORDS)

5'-Nucleotidase↗
PubMed1994

Morphology and distribution of ecto-5'-nucleotidase-positive cells in the rat choroid plexus.

The aim of this report was to find out whether adenosine can be produced locally in the choroid plexus of rats. Therefore we investigated the distribution of the enzyme ecto-5'-nucleotidase which hydrolyzes extracellular adenosine monophosphate to adenosine and phosphate. Enzyme activity histochemistry and immunohistochemistry demonstrated that ecto-5'-nucleotidase is present in the stroma but not in the epithelium. The positive cells in the stroma were identified as fibroblasts by their localization and by their shape. Double-labelling immunohistochemistry actually showed that ecto-5'-nucleotidase was absent from MHC class II-positive cells and from vessel walls. These data indicate that adenosine may be produced in the choroid plexus, and specifically in the interstitium. From there, adenosine would have direct access to nerves, immune cells, the epithelium and microvessels. Because adenosine has been reported to modulate blood supply and the rate of production of cerebrospinal fluid, a local control mechanism involving adenosine might operate in the choroid plexus in a similar way to that described in other tissues. Effects of adenosine on nerves and immune cells are discussed. The exclusive presence of ecto-5'-nucleotidase in the fibroblasts that are in contact with choroid plexus epithelium suggests that the expression of the enzyme is controlled by factors produced by epithelial cells, for instance by extracellular nucleotides.

5'-Nucleotidase↗
PubMed1994

Effect of dipyridamole on glomerular mesangial cell ecto-5'-nucleotidase expression.

Although dipyridamole has been extensively studied as an anti-aggregating agent, its mechanism of action has not been elucidated. Cultured mesangial cells were treated with dipyridamole 1-100 microM from 6-72 h. Ecto-5'-nucleotidase activity approximately doubled (from 115 +/- 11 to 226 +/- 14 nmol/min/mg) after treatment with 100 microM dipyridamole for 72 h. This effect was concentration- and time-dependent. Cycloheximide, an inhibitor of protein synthesis, did not alter basal 5'-nucleotidase activity. However, it prevented stimulation by 5 microM dipyridamole. Adenosine availability at the receptor sites was increased by dipyridamole and S-(p-nitrobenzyl)-6-thioinosine (NBTI), which inhibit adenosine uptake into the cell. Addition of dipyridamole or NBTI to the adenosine-treated mesangial cells produced an additive increase in ecto-5'-nucleotidase activity. Dipyridamole, through its effect on extracellular adenosine and ecto-5'-nucleotidase, may have an influence upon regulation of the glomerular microcirculation.

5'-Nucleotidase↗
PubMed1989

5-nucleotidase activity in cultured cell lines. Effect of different assay conditions and correlation with cell proliferation.

The 5-AMPase activity of the ectoenzyme 5-nucleotidase has been measured in a variety of cell lines, using intact cells. Human cell types showed two orders of magnitude higher enzyme activity than mouse cell lines. The ectoenzyme is inhibited by adenosine 5-(alpha, beta-methylene) diphosphate and Concanavalin A. A different extent of 5-nucleotidase lectin inhibition was observed in the studied cell lines, suggesting that the corresponding ectoenzymes are glycoproteins with a different type or degree, or both, of glycosylation. The 5-nucleotidase activity increased during subculture and decreased after cell transformation. Generally, the 5-nucleotidase activity was two- to five-fold higher in monolayer than in suspension cell culture. A relation between cell growth and 5-AMPase activity was also observed. Enzyme activity increased at the end of the lag phase (glioblastoma cells) or during the exponential phase (the other two cell lines). After confluence, the activity decreased to the initial or even lower range of activity. Observed activity variations with cell proliferation correlate with modifications of 5-AMPase activity during subculture.

5'-Nucleotidase↗
PubMed1998

NO and cGMP facilitate adenosine production in rat hearts via activation of ecto-5'-nucleotidase.

We examined whether nitric oxide (NO), a possible cardioprotective substance, can increase the production of interstitial adenosine in the ventricular myocardium. A flexibly mounted microdialysis technique was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase in in vivo rat hearts. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and perfused with Tyrode solution containing adenosine 5'-monophosphate (AMP) at a rate of 1.0 microl min-1. The concentration of adenosine in the effluent (dialysate) was measured by high-performance liquid chromatography. Dialysate adenosine obtained during perfusion with the AMP-containing solution through the probe originated from the hydrolysis of AMP by endogenous ecto-5'-nucleotidase, and the level of adenosine reflected the activity of ecto-5'-nucleotidase in the tissue. S-Nitroso-N-acetylpenicillamine (SNAP, 0.3-3 mM), an NO donor, increased the dialysate adenosine measured in the presence of AMP (100 microM) in a concentration-dependent manner. However, in the presence of an NO-oxidizing agent, 2-(4-carboxyphenyl-4,4,5, 5-tetramethylimidazoline)-1-oxyl 3-oxide (carboxy-PTIO, 1 mM), the effect of SNAP was abolished. Another NO donor, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409, 1 mM) also increased adenosine production. 8-Bromo-cGMP (0.1-3 mM), a membrane-permeable cGMP analogue and a potent activator of cGMP-dependent protein kinase, increased the level of AMP-primed dialysate adenosine in a concentration-dependent manner. These results suggest that NO facilitates the production of interstitial adenosine in rat hearts in situ, via cGMP-mediated activation of ecto-5'-nucleotidase.

5'-Nucleotidase↗
PubMed1976

Effect of sodium deoxycholate on 5'-nucleotidase.

The 5'-nucleotidase localized in rat liver plasma membranes was purified to a single protein, which contained phospholipid. The molecular weight and the sedimentation constant were about 150 000 and 7 S in the presence of sodium deoxycholate, while the enzyme protein was aggregated when the preparation was dialyzed thoroughly. The purified 5'-nucleotidase exhibited the same properties as the 5'-nucleotidase in plasma membranes. The 5'-nucleotidase activity was increased by the addition of various bile salts or by the solubilization of membranes with trypsin, papain or phospholipase C. The solubilized and aggregated forms of the enzyme showed different substrate specificity for nucleotides, pH optimum, heat stability and Km. The purified enzyme catalyzed an exchange reaction between AMP and adenosine, which was diminished by the addition of sodium deoxycholate.

Adenosine Monophosphate↗
PubMed1990

Characterization of soluble vs membrane-bound human placental 5'-nucleotidase.

Three forms of 5'-nucleotidase purified from human placenta (two membrane-bound forms, one sensitive and one resistant to cleavage by phosphatidylinositol-specific phospholipase C, as well as a soluble form) had the same molecular weight before (73,000 Da) and after (56,000 Da) digestion with N-glycosidase F and showed similar amino acid compositions, N-terminal amino acid sequences, and KMs for IMP (9.6 to 11.9 microM). Thus, these three forms of 5'-nucleotidase appear to have very similar structures. The form sensitive to phosphatidylinositol-specific phospholipase C contained nearly 1 mol myo-inositol/mol of protein as determined by mass spectrometry, indicating a glycosyl phosphatidylinositol membrane anchor. Soluble 5'-nucleotidase contained a similar quantity of myo-inositol, suggesting that it was previously membrane-anchored via glycosyl phosphatidylinositol. The form resistant to phosphatidylinositol-specific phospholipase C contained less myo-inositol, leaving open the possibility of a third form of 5'-nucleotidase with a conventional transmembrane anchor.

5'-Nucleotidase↗
PubMed1991

Electrophoretic separation of 5'-nucleotidase multiple forms.

A procedure is proposed for the separation of multiple forms of 5'-nucleotidase (EC 3.1.3.5) by cellulose acetate electrophoresis. The effects of various treatments (wheat-germ lectin, neuraminidase, n-butanol, papain, Triton X-100 and precipitation of LDL and VLDL) on the electrophoretic pattern of 5'-nucleotidase and alkaline phosphatase were studied. In healthy controls, the presence of three fractions with alpha 1, alpha 2 and beta mobilities was observed, the latter being the major one. In different hepatobiliary diseases a close relationship between the presence of high molecular weight alkaline phosphatase and the increase in the ratio Total/beta 5'-nucleotidase was observed, showing that the increase in total 5'-nucleotidase in these patients is mainly due to the alpha 1 isoform. The nature of these forms is discussed.

5'-Nucleotidase↗
PubMed1990

Soluble "high Km" 5'-nucleotidase activity in human T- and B-lymphoblasts: isolation and some properties.

1. Activity of "high Km" 5'-nucleotidase was investigated in the soluble fractions from cultured human T- and B-lymphoblasts. 2. Using gel filtration chromatography and 5'-AMP-Sepharose 4B affinity chromatography, it separated high Km 5'-nucleotidases from other two different soluble nucleoside 5'-phosphomonoesterase activities. 3. The molecular mass of the high Km enzymes from T- and B-lymphoblasts were 210 and 200 kDa, respectively. The optimum pH was at 6.5, and the Km values for IMP and AMP were 0.4 and 0.9 mM, respectively. 4. These properties of high Km 5'-nucleotidases were similar to those previously described from different tissues. These data indicate that soluble high Km 5'-nucleotidase coexists with "low Km" enzyme.

5'-Nucleotidase↗