Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “NUCLEOTIDASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed2006

Expression of ecto-5'-nucleotidase (eN, CD73) in cell lines from various stages of human melanoma.

Ecto-5'-nucleotidase is a GPI-anchored enzyme localized in cell membrane lipid rafts. Although it is highly expressed in many tumour cells, its specific function during tumorigenesis is unclear. We have found that, among different melanoma cells, upregulated expression of ecto-5'-nucleotidase is associated with a highly invasive phenotype. Analysis of other cell membrane proteins involved in melanoma adhesion and metastasis demonstrated that expression of alpha5, beta1, beta3-integrin subunits and CD44 was elevated gradually in accordance with increasing metastatic potential. Expression of alphav-integrin and caveolin-1 was seen mostly in cells derived from metastatic melanomas. Furthermore, in contrast to N-cadherin, which was unaltered in all lines, we could not detect E-cadherin in any cell type. Functional assays demonstrated that highly expressed ecto-5'-nucleotidase is a catalytically competent protein that is very sensitive to inhibition by concanavalin A. The interaction with concanavalin A also caused increased association of ecto-5'-nucleotidase-rich lipid rafts with much heavier cytoskeletal complexes as determined by density gradient centrifugation. A similar shift towards heavier cytoskeletal fractions also took place with other proteins coexpressed with ecto-5'-nucleotidase, such as alphav, alpha5, beta1 and beta3-integrins, caveolin-1 and CD44. As ConA-induced clustering may reflect the interactions of membrane proteins with extracellular matrix, we also analysed the effect of several extracellular matrix proteins on the in-situ activity of ecto-5'-nucleotidase in WM9 cells and found that tenascin C strongly inhibited ecto-5'-nucleotidase activity and adenosine generation from AMP. We also developed WM9 cells with reduced ecto-5'-nucleotidase expression and tested differences in cell adhesion on various extracellular matrix proteins. WM9 cells attached significantly weaker to tenascin C layer. These observations indicate that expression of ecto-5'-nucleotidase correlates with a number of metastasis-related markers and thus may have a function in this process. Furthermore, our data suggest that, in addition to generating adenosine, ecto-5'-nucleotidase may have independent roles in adhesion and interaction with extracellular matrix components in melanoma.

5'-Nucleotidase↗
PubMed1995

Alpha 1-adrenoceptor activation increases ecto-5'-nucleotidase activity and adenosine release in rat cardiomyocytes by activating protein kinase C.

BACKGROUND: Adenosine is an important regulator of many cardiac functions and is synthesized primarily by ecto- and cytosolic 5'-nucleotidase. We have previously reported that alpha 1-adrenoceptor blockade attenuates adenosine release from ischemic myocardium, raising the possibility that alpha 1-adrenoceptor activation activates 5'-nucleotidase. This study tested whether activation of protein kinase C by alpha 1-adrenoceptor activation increases 5'-nucleotidase activity and augments adenosine release. METHODS AND RESULTS: Cardiomyocytes were isolated from adult male Wistar rats and suspended in modified HEPES-Tyrode's buffer solution. After stabilization, the cardiomyocytes were incubated with and without an exposure to norepinephrine (10(-9) to 10(-5) mol/L) while being treated with propranolol and yohimbine or with and without an exposure to methoxamine (10(-9) to 10(-5) mol/L). Ecto-5'-nucleotidase activity was increased by norepinephrine and methoxamine during 30 minutes in a dose-dependent manner, whereas cytosolic 5'-nucleotidase was not activated. These increases in ecto-5'-nucleotidase activity were inhibited by GF109203X, an inhibitor of protein kinase C, and mimicked by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. The increase in ecto-5'-nucleotidase was not prevented by cycloheximide. When ecto-5'-nucleotidase activity increased, adenosine release was augmented in methoxamine- and PMA-treated cardiomyocytes (1299 +/- 252% and 1372 +/- 149%, respectively) compared with the untreated group (578 +/- 26%). The increase in adenosine release was blunted by GF109203X and alpha, beta-methyleneadenosine 5'-diphosphate, an inhibitor of ecto-5'-nucleotidase. CONCLUSIONS: Thus, we conclude that alpha 1-adrenoceptor-mediated increases in ecto-5'-nucleotidase activity are attributed to activation of protein kinase C in rat cardiomyocytes.

5'-Nucleotidase↗
PubMed2003

Hypoxia enhances Ecto-5'-Nucleotidase activity and cell surface expression in endothelial cells: role of membrane lipids.

Extracellular adenosine production by the glycosyl-phosphatidyl-inositol-anchored Ecto-5'-Nucleotidase plays an important role in the defense against hypoxia, particularly in the intravascular space. The present study was designed in order to elucidate the mechanisms underlying hypoxia-induced stimulation of Ecto-5'-Nucleotidase in endothelial cells. For this purpose, aortic endothelial cells (SVARECs) were submitted to hypoxic gas mixture. Hypoxia (0% O2 for 18 hours) induced a 2-fold increase of Ecto-5'-Nucleotidase activity (Vmax 19.78+/-0.53 versus 8.82+/-1.12 nmol/mg protein per min), whereas mRNA abundance and total amount of the protein were unmodified. By contrast, hypoxia enhanced cell surface expression of Ecto-5'-Nucleotidase, as evidenced both by biotinylation and immunostaining. This effect was accompanied by a decrease of Ecto-5'-Nucleotidase endocytosis, without modification of Ecto-5'-Nucleotidase association with detergent-resistant membranes. Finally, whereas cholesterol content was unmodified, hypoxia induced a time-dependent increase of saturated fatty acids in SVARECs, which was reversed by reoxygenation, in parallel to Ecto-5'-Nucleotidase stimulation. Incubation of normoxic cells with palmitic acid enhanced Ecto-5'-Nucleotidase activity and cell surface expression. In conclusion, hypoxia enhances cell surface expression of Ecto-5'-Nucleotidase in endothelial cells. This effect could be supported by a decrease of Ecto-5'-Nucleotidase endocytosis through modification of plasma membrane fatty acid composition.

5'-Nucleotidase↗
PubMed1993

Dual mechanism of laminin modulation of ecto-5'-nucleotidase activity.

The myoblast cell surface activity of ecto-5'-nucleotidase was stimulated by a laminin substrate, whereas fibronectin and gelatin did not increase the AMPase activity of ecto-5'-nucleotidase. This increase was related to a higher expression of ecto-5'-nucleotidase on the surface of cells seeded on a laminin substrate, but without the mobilization of an intracellular pool of enzyme. Furthermore, laminin and its fragments E'1 and E8 modified the AMPase activity of the ecto-5'-nucleotidase purified from chicken striated muscle and reconstituted in liposomes. Over the range of concentrations used, intact laminin and its fragment E8, consisting of the distal half of the long arm, stimulated the AMPase activity of ecto-5'-nucleotidase. By contrast, the large fragment derived from the short arms, designated E'1, inhibited the AMPase activity. Furthermore, the monoclonal anti-ecto-5'-nucleotidase antibody, CG37, abolished the stimulatory effect of fragment E8 on the AMPase activity of ecto-5'-nucleotidase but did not reverse the inhibitory effect of fragment E'1. In conclusion, laminin stimulates the AMPase activity of ecto-5'-nucleotidase by two mechanisms: inducing the expression of ecto-5'-nucleotidase to the cell surface and direct modulation of the enzymatic activity.

5'-Nucleotidase↗
PubMed1986

Antibody against mouse liver 5'-nucleotidase immunostains white matter in the adult mouse central nervous system.

An antiserum against rat liver 5'-nucleotidase has been shown to immunostain myelinated fibers and oligodendrocytes in the rat CNS, consistent with evidence for 5'-nucleotidase activity in rat brain myelin and oligodendrocytes (Cammer, Sacchi and Kahn, Devel. Brain Res., 1985, 20: 89-96). However, in the mouse CNS, in which myelin also has 5'-nucleotidase activity, that antiserum stained only blood vessels. To obtain an antibody against the mouse enzyme, 5'-nucleotidase was partly purified from mouse liver membranes by detergent extraction, heat treatment, affinity chromatography, acidification, and ammonium sulfate fractionation. The preparation, which was enriched about 110-fold in 5'-nucleotidase specific activity, compared to the starting extract, was electrophoresed on a preparative slab gel containing Triton X-100, a strip was stained histochemically for 5'-nucleotidase, and the material corresponding to the stained band was used to immunize a rabbit. The immune IgG fraction, but not the preimmune IgG, reacted with mouse brain homogenates. The immune serum gave consistently greater inhibition of 5'-nucleotidase activity in mouse liver homogenates, mouse brain myelin and mouse brain homogenates, but not rat brain or liver homogenates, compared to the preimmune serum. The immune serum, but not the preimmune serum, immunostained white matter in the normal adult mouse brain and spinal cord. The findings suggest that the mouse may have one isozyme of 5'-nucleotidase similar to that in rat with respect to subunit sizes but differing in primary structure at one or more antigenic sites and support previous observations of 5'-nucleotidase activity in myelin from mouse brains and spinal cords.

5'-Nucleotidase↗
PubMed1988

Monospecific antiserum against 5'-nucleotidase from Torpedo electric organ: immunocytochemical distribution of the enzyme and its association with Schwann cell membranes.

The cellular and subcellular distribution of 5'-nucleotidase in tissues of the electric ray Torpedo marmorata has been investigated by means of an antiserum raised against the native enzyme purified from the electric organ. As revealed by immunohistochemistry the enzyme is associated with the surface of the axons of the electric nerves and of spinal nerves. Using the post-embedding colloidal gold technique at the electron-microscopical level 5'-nucleotidase could be located at the plasma membrane of the Schwann cells including the myelin and the fine processes covering the terminal axon ramifications. Also the perineurial sheath of the axons inside the electric organ is 5'-nucleotidase positive. The plasma membrane of the axon and the terminal axon region or the postsynaptic membrane do not contain 5'-nucleotidase. Immunoprecipitation studies using polyacrylamide beads suggest that the ecto-Ca2+- or -Mg2+-adenosine 5'-triphosphatase previously ascribed to synaptosomes of the Torpedo electric organ is not associated with the same membranes as 5'-nucleotidase. Within the electric organ the dorsal plasma membrane of the electroplaque cell, blood capillaries and the connective tissue layer surrounding the columns of electroplaque cells also bind the antibodies. In central nervous tissue solely blood vessels show immunofluorescence. Within the electric lobe both the surface of the electromotor neurons as well as the myelinated axons giving rise to the electric nerve are negative. This also applies to the axons of the optic nerve suggesting that the antiserum is Schwann cell specific, and does not bind to a potential oligodendroglial 5'-nucleotidase. In peripheral tissue the surface of skeletal muscle fibres as well as that of individual myofibrils bind the anti-5'-nucleotidase antibodies. Our results demonstrate that the Schwann cell plasma membrane, including myelin, contains 5'-nucleotidase and that one can distinguish by means of a specific antiserum between Schwann cell and oligodendroglia plasma membranes. The functional significance of the association of 5'-nucleotidase with Schwann cells along the entire surface of axons including the synaptic region as well as with other parts of the electric tissue is discussed regarding its catalytic activity and also the possibility that this surface glycoprotein may be involved in mediating cellular interactions.

5'-Nucleotidase↗
PubMed1982

Regulation of purine metabolism by plasma membrane and cytoplasmic 5'-nucleotidases.

The contribution of plasma membrane 5'-nucleotidase (E.C. 3.1.3.5) to intracellular purine degradation and release was evaluated in cultured human lymphoblasts. B-lymphoblasts and T-lymphoblasts are characterized by high and low levels of plasma membrane 5'-nucleotidase activity, respectively. After radiolabeling of the cellular adenine nucleotide pools with [8-14C]adenine, deoxyglucose-induced purine nucleotide degradation resulted in a 2-2.5 times greater release of cellular radioactivity from the B-lymphoblasts than from the T-lymphoblasts. Specific inhibition of plasma membrane 5'-nucleotidase with 50 microM alpha, beta-methylene adenosine diphosphate (AMPCP) did not decrease purine release during deoxyglucose-induced nucleotide degradation. Similarly, the inhibition of B-lymphoblast membrane 5-nucleotidase did not alter the incorporation of [8-14C]adenine into the nucleotide pool. Therefore, to explain the relatively high release of purine nucleotide degradation products in B-lymphoblasts when compared with T-lymphoblasts, cytoplasmic 5'-nucleotidase activity was investigated in these cell lines. B-lymphoblasts have seven times more cytoplasmic 5'-nucleotidase activity for dAMP and two to three times more activity for other purine nucleoside 5'-monophosphates than do T-lymphoblasts at pH 7.4. Membrane and cytoplasmic nucleotidase activities are produced by different enzymes that can be distinguished by differences in pH optima, Michaelis constants for purine substrates, divalent cation requirements, and susceptibilities to AMPCP inhibition. The data suggest that plasma membrane 5'-nucleotidase hydrolyzes extracellular nucleoside 5'-monophosphates only. Cytoplasmic 5'-nucleotidase most likely regulates the degradation of intracellular nucleoside 5'-monophosphates and may be responsible for the increased purine release observed in B-lymphoblasts.

5'-Nucleotidase↗
PubMed1992

5'-nucleotidase: a new marker for striosomal organization in the rat caudoputamen.

The distribution of the adenosine-producing ectoenzyme 5'-nucleotidase was studied by means of a histochemical lead technique in the caudoputamen of normal adult rats and of rats in which injections either of 6-hydroxydopamine in the medial forebrain bundle or of ibotenic acid in the caudoputamen had been made 1-3 weeks previously. The patterns of striatal 5'-nucleotidase activity in these animals were compared in serial sections to the patterns of calbindin-D28k immunoreactivity and of 3H-naloxone ligand binding, which respectively mark the known matrix and striosome (patch) compartments of the caudoputamen. In the normal rats, 5'-nucleotidase activity was differentially concentrated in striosomes, where it produced a dense staining of the neuropil. The enzymatic staining followed a striosomal distribution in all but the caudal caudoputamen. Within the striatal matrix, 5'-nucleotidase staining also observed a lateromedial density gradient. Depletion of the dopamine-containing nigrostriatal innervation of the caudoputamen with 6-hydroxydopamine did not alter the striosomal selectivity of 5'-nucleotidase activity. Destruction of intrastriatal neurons by ibotenic acid led to a strongly 5'-nucleotidase-positive gliosis within the resulting necrotic region. Elsewhere in the caudoputamen, the enzyme's striosomal distribution was not detectably altered. We conclude that 5'-nucleotidase histochemistry provides an advantageous tool for detecting the striosomal architecture of the rat's caudoputamen. Moreover, 5'-nucleotidase is prominently associated with glial membranes in the central nervous system, so that the concentration of this enzyme in striosomes could mark these as sites of selective glial populations within striatum. These properties and actions of 5'-nucleotidase in purinergic neurotransmission and in neuroadhesion may contribute to the specialized functions of striosomes and matrix.

5'-Nucleotidase↗
PubMed1994

Synaptic 5'-nucleotidase activity reflects lesion-induced sprouting within the adult rat dentate gyrus.

In development, the ectoenzyme 5'-nucleotidase marks maturing cerebellar and cortical synapses, but it is localized in glial cells in the normal, adult nervous system. With a histochemical lead technique, we have now investigated its localization during reactive synaptogenesis in the dentate gyrus of adult rats deprived of entorhinal afferents. A band of enhanced 5'-nucleotidase reaction product was present in the outer portions of the dentate molecular layer between 5 and 75 days after destruction of the ipsilateral entorhinal cortex. At the ultrastructural level, 5'-nucleotidase-positive microglia and degenerating axon terminals were numerous within this band during the first postoperative week. Between Days 7 and 75, intact synapses were found that exhibited 5'-nucleotidase reaction product in their clefts. Astrocytic labeling was abundant. No enzyme-positive synapses and few labeled glial elements were present in the control molecular layer. Conspicuous 5'-nucleotidase activity within synaptic clefts of mossy fiber terminals was present between Postoperative Days 10 and 40 on the operated side, but the staining was sporadic on the control side. We conclude that 5'-nucleotidase is associated with lesion-induced synaptic remodeling in the dentate gyrus. The band of 5'-nucleotidase reaction product within the outer molecular layer corresponds to the zone where the lesioned entorhinal fibers degenerate and where other afferents sprout. Here, the transient appearance of 5'-nucleotidase within synaptic clefts parallels the time course of synaptic reinnervation. The enzyme is also indicative of the sprouting response of mossy fiber terminals. Functional properties of 5'-nucleotidase in purinergic neuromodulation and cellular adhesion may be relevant for the generation and plasticity of synaptic contacts.

5'-Nucleotidase↗
PubMed1991

Cyclic adenosine monophosphate-stimulating agents induce ecto-5'-nucleotidase activity and inhibit DNA synthesis in rat cultured mesangial cells.

The ecto-5'-nucleotidase activity of rat glomerular mesangial cells increases after exposure to prostaglandin E2 (PGE2) via cAMP stimulation (Savic et al., 1990, Immunology 70, 321). Therefore we examined whether other cAMP-stimulating agents had a similar effect. Forskolin (1 microM), PGE2 (10 microM), and isoproterenol (10 microM), three products stimulating rat mesangial cell adenylate cyclase activity, enhanced cAMP accumulation within 5 min and 5'-nucleotidase activity after a lag time of at least 24 h, 3-Isobutyl-1-methylxanthine (IBMX) and Ro 20-1724, two drugs inhibiting cAMP degradation, also stimulated cAMP accumulation and 5'-nucleotidase activity. The effects of these agents on 5'-nucleotidase activity were additive with those of the three products stimulating adenylate cyclase activity, except for Ro 20-1724 and forskolin which acted synergistically. Cycloheximide, a blocker of protein synthesis, suppressed the cAMP-dependent increase of 5'-nucleotidase activity. Because ecto-5'-nucleotidase activity is a marker of cell differentiation, the effect of the same cAMP-stimulating agents on cell proliferation was also studied. Forskolin, PGE2, and isoproterenol inhibited [3H]thymidine incorporation into rat mesangial cells in a dose-dependent manner. The same effect was obtained with IBMX (100 microM) and Ro 20-1724 (50 microM). Stimulation of 5'-nucleotidase activity and inhibition of [3H]thymidine incorporation occurred over the same range of concentrations for the various agonists tested. Taken together, these results indicate that expression of ecto-5'-nucleotidase in rat mesangial cells is induced by cAMP whatever the reason for its accumulation. The simultaneous inhibition of DNA synthesis may occur independently or be associated with the stimulation of 5'-nucleotidase expression.

5'-Nucleotidase↗
PubMed1992

Limited proteolysis of chicken gizzard 5'-nucleotidase.

Chicken gizzard 5'-nucleotidase represents an ectoenzyme which is linked to the plasma membrane via a phosphatidylinositol glycan. We have characterized the possible domain-like organization of 5'-nucleotidase by limited proteolysis. A hydrophobic proteolytic fragment carrying the intact C-terminus, as well as two major hydrophilic products, were identified. We developed procedures for specific radiolabelling of the active center of 5'-nucleotidase. This allowed us to locate the catalytic site within hydrophilic fragments obtained after limited proteolysis. We demonstrate that removal of N-linked carbohydrate chains increases the sensitivity of 5'-nucleotidase to proteolytic attack, indicating that sugar moieties protect against proteolysis. 5'-Nucleotidase represents a binding protein for components of the extracellular matrix. The interaction between 5'-nucleotidase and the laminin/nidogen complex unmasked proteolytic cleavage sites in the C-terminal portion of the enzyme. This resulted in the specific production of a hydrophilic form of 5'-nucleotidase. In summary, we have further characterized chicken gizzard 5'-nucleotidase: (1) the protein is organized into two domain-like structures, (2) the N-terminal domain harbors the active center; (3) N-linked carbohydrates protect the protein against proteolytic degradation; (4) interaction with components of the extracellular matrix alters the conformation of 5'-nucleotidase.

5'-Nucleotidase↗
PubMed1992

Application of solid-phase extraction on anion-exchange cartridges to quantify 5'-nucleotidase activity.

The enzyme 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) catalyzes a critical reaction in intermediary metabolism, the phosphohydrolysis of nucleoside 5'-monophosphates to their corresponding nucleosides. We have evaluated solid-phase extraction on pre-packed anion-exchange cartridges as a chromatographic technique with which 5'-nucleotidase activity may be detected and quantified. Chromatographic conditions were established whereby substrate nucleotide was rapidly and completely separated from its corresponding nucleoside by solid-phase extraction. Both analytes were recovered quantitatively, without loss or degradation. This chromatographic system was integrated into a discontinuous radiochemical assay for 5'-nucleotidase which enabled both substrate utilization and product formation to be assessed simultaneously. Enzyme reaction samples could be analyzed directly for 5'-nucleotidase activity without any pre-chromatography preparation. The high capacity of the solid-phase cartridges and the inability of 5'-nucleotidase to enter the packing bed during analyte elution facilitated termination of the enzyme reaction by applying the entire reaction mixture to the cartridge. Loaded cartridges could then be stored at 4 degrees C prior to chromatography and subsequently batch-eluted. The excellent resolution between substrate and product in solid-phase extraction and the sensitivity of radioisotopic counting enabled detection/quantification of low tissue levels of 5'-nucleotidase in conjunction with ancillary assays for secondary enzyme reactions with the potential to elicit the artifactual loss of 5'-nucleotidase substrate/product when crude biological preparations are examined for 5'-nucleotidase activity. Our results demonstrate that solid-phase extraction on anion-exchange cartridges with elution solvents of appropriate pH offers several unique advantages for 5'-nucleotidase determination.

5'-Nucleotidase↗
PubMed1998

5'-nucleotidase activity in lymphocytes from patients affected by B-cell chronic lymphocytic leukemia.

OBJECTIVES: The activity of membrane-bound ecto-5'-nucleotidase and soluble e-Ns and c-N-II 5'-nucleotidases was evaluated on lymphocytes from patients affected by B-cell chronic lymphocytic leukemia (B-CLL). A statistically significative decrease in ecto-5'-nucleotidase, e-Ns, and c-N-II activities was observed in peripheral blood lymphocytes and in B and T populations from affected individuals. DESIGN AND METHODS: For the assay of ecto-5'-nucleotidase, e-Ns, and c-N-II activity we used a radioactive procedure coupled to HPLC. Since the ecto-5'-nucleotidase is identified as CD73 antigen, we performed immunofluorescence analysis using a specific monoclonal antibody. We analyzed ecto-5'-nucleotidase mRNA by RT-PCR to ascertain the possibility of an alteration in the transcription of its gene. RESULTS: A decrease in ecto-5'-nucleotidase activity was correlated to reduction in ecto-5'-nucleotidase positive cells (CD73+) in leukemia patients. RT-PCR produced a fragment of the expected size and the specific mRNA was found expressed in both healthy subjects and leukemia patients. CONCLUSIONS: The decrease in ecto-5'-nucleotidase activity in patients with B-CLL is not due to loss of transcription of the specific mRNA. The presence of point mutations, splicing alteration, or posttranslational modifications must be investigated. If a defect at DNA or RNA level will be detected, the molecular analysis will be considered for diagnosis of B-cell chronic lymphocytic leukemia.

5'-Nucleotidase↗
PubMed1993

Attenuation of ecto-5'-nucleotidase activity and adenosine release in activated human polymorphonuclear leukocytes.

To examine whether activation of polymorphonuclear leukocytes attenuates release of adenosine through attenuation of their own ecto-5'-nucleotidase activity, human polymorphonuclear leukocytes were incubated with and without exposure to either N-formyl-methionyl-leucyl-phenylalanine (FMLP) or complement C5a. Ecto-5'-nucleotidase activity of polymorphonuclear leukocytes was attenuated by both FMLP and complement C5a (22.7 +/- 3.6 vs 9.7 +/- 2.6 nmol/min per 10(7) cells at 10(-6) M FMLP, P < .05; 21.5 +/- 2.2 vs 10.2 +/- 1.2 nmol/min per 10(7) cells at 5 x 10(-7) g/mL complement C5a, P < .001), whereas cytosolic 5'-nucleotidase activity was not affected by either FMLP or complement C5a. These reductions of ecto-5'-nucleotidase activity that were caused by both FMLP and complement C5a were dose and time dependent and were inhibited by superoxide dismutase. Desferrioxamine did not inhibit the decreases in ecto-5'-nucleotidase. In accordance with the decreases in ecto-5'-nucleotidase activity, release of adenosine was attenuated in the FMLP-pretreated and complement C5a-pretreated polymorphonuclear leukocytes, which were restored by concomitant administration of superoxide dismutase. The viability of FMLP-pretreated and complement C5a-pretreated polymorphonuclear leukocytes was markedly decreased compared with the untreated group after 60 minutes of hypoxia followed by 60 minutes of reoxygenation. Thus, we conclude that: (1) activation of polymorphonuclear leukocytes attenuates their own ecto-5'-nucleotidase activity and thereby reduces adenosine release, (2) reduction of ecto-5'-nucleotidase activity is attributable to generated superoxide anion in polymorphonuclear leukocytes, and (3) viability of polymorphonuclear leukocytes after hypoxia and reoxygenation largely depends on the extents of decreases in ecto-5'-nucleotidase activity.

5'-Nucleotidase↗
PubMed1994

Evidence for deactivation of both ectosolic and cytosolic 5'-nucleotidase by adenosine A1 receptor activation in the rat cardiomyocytes.

Adenosine, an important regulator of many cardiac functions, is produced by ectosolic and cytosolic 5'-nucleotidase. The activity of these enzymes is influenced by several ischemia-sensitive metabolic factors, e.g., ATP, ADP, H+, and inorganic phosphate. However, there is no clear evidence that adenosine itself affects 5'-nucleotidase activity. This study tested whether adenosine decreases the activity of ectosolic and cytosolic 5'-nucleotidase. Cardiomyocytes were isolated from adult male Wistar rats and suspended in the modified Hepes-Tyrode buffer solution. After stabilization, isolated cardiomyocytes were incubated with and without adenosine (10(-9) - 10(-4) M). Ectosolic and cytosolic 5'-nucleotidase activity was decreased by exogenous adenosine (ectosolic 5'-nucleotidase activity, 20.6 +/- 2.3 vs. 8.6 +/- 1.6 mumol/min per 10(6) cells [P < 0.05]; cytosolic 5'-nucleotidase activity, 2.47 +/- 0.58 vs. 1.61 +/- 0.54 mumol/min per 10(6) cells [P < 0.05] at 10(-6) M adenosine) after 30 min. The decrease in ectosolic and cytosolic 5'-nucleotidase activity was inhibited by 8-phenyltheophylline and pertussis toxin, and was mimicked by N6-cyclohexyladenosine, an adenosine A1 receptor agonist. Neither CGS21680C, and A2 receptor agonist, nor cycloheximide deactivated ectosolic and cytosolic 5'-nucleotidase. Thus, we conclude that activation of adenosine A1 receptors is coupled to Gi proteins and attenuates ectosolic and cytosolic 5'-nucleotidase activity in rat cardiomyocytes.

5'-Nucleotidase↗
PubMed1992

Chicken gizzard 5'-nucleotidase functions as a binding protein for the laminin/nidogen complex.

Soluble and reconstituted 5'-nucleotidase were used in the binding assays to the laminin/nidogen complex. They both are shown to interact specifically and in a saturable manner with the laminin/nidogen complex using a solid-phase binding assay. Dissociation constants in the region of 10(-8) M were determined for the association of soluble and membrane-bound 5'-nucleotidase. Scatchard analysis of the binding data indicate a stoichiometry of about 2.7 of the homodimeric soluble 5'-nucleotidase to the laminin/nidogen complex. The association of 5'-nucleotidase with laminin/nidogen occurs in the absence of divalent metal ions and does not require N-linked carbohydrate moieties of both laminin/nidogen and 5'-nucleotidase. 5'-Nucleotidase also associates with isolated laminin although with reduced affinity. No binding to isolated nidogen was observed. Peptides containing the RGD sequence did not influence the binding reaction. Monoclonal and polyclonal antibodies directed against 5'-nucleotidase and laminin specifically perturb the association of the reconstituted enzyme to laminin/nidogen. Sulfated polysaccharides such as heparinsulfate and dermatansulfate modulate the interaction of 5'-nucleotidase and laminin/nidogen in a complex biphasic manner and might also regulate the binding reaction in vivo. Immunohistochemistry shows a close spatial correlation of 5'-nucleotidase and laminin also in the epithelium of the small intestine pointing to an in vivo interaction of both glycoproteins.

5'-Nucleotidase↗
PubMed1996

Immunocytochemical localization of ecto-5'-nucleotidase in cultures of cerebellar granule cells.

The distribution of ecto-5'-nucleotidase, a glycosyl phosphatidylinositol anchored membrane protein capable of hydrolysing extracellular nucleoside monophosphates, was investigated by immunocytochemistry in cultures of rat cerebellar cells obtained at postnatal days 6 and 8. The enzyme was expressed at the surface of granule cells including their neurites as well as on other neurons in the culture. The distribution of 5'-nucleotidase matched that of the synaptic vesicle protein 2. Oligodendroglial cells were identified by their immunoreactivity for 2',3'-cyclic nucleotide 3'-phosphodiesterase. Their entire surface was labelled for 5'-nucleotidase. In contrast, only a subset of astrocytes immunopositive for the glial fibrillary acidic protein revealed surface-located immunoreactivity for 5'-nucleotidase. Antibody-binding of the labelled-astrocytes was enhanced at restricted surface domains. Endothelial cells that avidly bind Lycopersicon esculentum lectin were the most intensely anti-5'-nucleotidase-labelled cell type of the culture. Double labelling revealed an exact match of surface-located antibody binding sites for 5'-nucleotidase and laminin. Immunoreactivity for 5'-nucleotidase was essentially absent from fibroblasts that could be identified by their immunoreactivity for fibronectin. All cell types that carried surface-bound 5'-nucleotidase also revealed a cytoplasmic pool of the enzyme. Our results provide the first immunocytochemical demonstration of the surface-location of 5'-nucleotidase in neurons. They suggest that the broad distribution of the enzyme at the surface of neurons and other cells types from neonatal brain reflects its functional importance in the differentiation of the nervous system.

Adenosine Triphosphatases↗
PubMed1981

Rat brain 5'-nucleotidase: developmental changes in myelin and activities in subcellular fractions and myelin subfractions.

The activities of 5'-nucleotidase, measured in brain homogenates and myelin isolated from rats at 21, 60 and greater than 90 days of age, were compared to values for two other myelin-associated enzymes, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and carbonic anhydrase. Whereas the activities of all 3 enzymes were higher in brain homogenates from 60-day-old rats than in those from 21-day-old rats, only 5'-nucleotidase increased significantly in specific activity in both homogenates and myelin after the age of 60 days. The ratios of 5'-nucleotidase to the myelin basic and proteolipid proteins in subcellular fractions from adult rat brain suggested that the microsomal fraction was the only fraction containing 5'-nucleotidase levels not attributable to contamination by myelin membranes. Like carbonic anhydrase, 5'-nucleotidase had a greater distribution than CNP into microsomes of adult rats. When purified myelin was fractionated on a density gradient, the specific activity of 5'-nucleotidase was highest in the heaviest subfraction, with recovery of significant activity occurring, however, in all 3 subfractions. In rats over 60 days of age the recovery of 5'-nucleotidase in myelin was almost as high as that of the relatively myelin-specific enzyme CNP, suggesting that myelin may be the predominant, although not exclusive locus of 5'-nucleotidase in the adult rat brain.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗