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PubMed1997

Overexpression of ecto-5'-nucleotidase promotes P-glycoprotein expression in renal epithelial cells.

UNLABELLED: P-glycoprotein (P-gp), responsible for multidrug resistance (MDR) of tumoral cells, is also expressed in apical membranes of normal epithelial cells, among which are proximal tubular cells. Ecto-5'-nucleotidase (5'Nu), co-located with P-gp in renal brush border membranes, could be instrumental in the expression of MDR phenotype. P-gp activity [assessed by rhodamine 123 (R123) and [3H]vinblastine (3H-VBL) accumulation] was evaluated in MDCK cell lines in which human 5'Nu was expressed at different levels after retroviral infection: MDCK-5'NU/- cells with a low 5'Nu activity (Vmax < 2 pmol/mg protein/min) and MDCK-5'NU/+ cells, which expressed a high level of 5'Nu (Vmax 150 +/- 18.5 pmol/mg protein/min). MDCK-5'NU/- cells did not display functional expression of MDR. In MDCK-5'NU/+ cells, R123 and 3H-VBL accumulation was significantly lower than in MDCK-5'NU/- cells and was dramatically enhanced by P-gp inhibitors. This high P-gp activity in MDCK-5'NU/+ cells was confirmed by their resistance to colchicine (measured by LDH release and MTT assay) as compared to MDCK-5'NU/- and was accounted for by increased membrane expression of P-gp assessed by Western blot. Neither AMP nor adenosine, the substrate and the product of 5'Nu, respectively, affected P-gp activity. Inhibition of 5'Nu with alpha beta-methylene-adenosine-diphosphate (alpha beta MADP) or with a blocking anti-5'Nu antibody (1E9) did not blunt MDR expression in MDCK-5'NU/+ cells. Conversely, the anti-5'Nu antibody 5F/F9, which did not block the enzymatic site, induced a decrease of P-gp activity. Further, incubation of MDCK-5'NU/- cells with conditioned medium from MDCK-5'NU/+ cells, which contained significant amounts of released 5'Nu, induced MDR phenotype. IN CONCLUSION: (i) expression of ecto-5'Nu promotes multidrug resistance (MDR) activity in renal epithelial cells by enhancement of P-gp expression; (ii) this effect does not involve enzymatic activity of 5'Nu; (iii) supernatants of cells that express 5'Nu conferred P-gp activity to 5'Nu negative cells.

5'-Nucleotidase↗

PubMed1997

Immunohistochemical colocalization of the alpha-subunit of neutrophil NADPH oxidase and ecto-5'-nucleotidase in kidney and liver.

In kidney and liver, fibroblasts and fibroblast-like cells, respectively, are sources of erythropoietin (Epo) formation, and these cells also bear a number of other similarities. Renal Epo expression is localized in peritubular type 1 fibroblasts of the cortical labyrinth, and in the liver, apart from parenchymal cells, transcription is found in Ito cells. Both the renal peritubular cells and Ito cells contain ecto-5'-nucleotidase (5'NT). It had been suggested that 5'NT is involved in the oxygen sensing mechanism via a hydrolysis of AMP to adenosine, which in turn may stimulate EPO synthesis. However, the molecular mechanism of the cellular response to hypoxia is currently not well understood. Based on the notion that a heme protein probably acts as the oxygen sensor, it has recently been proposed that a b-type cytochrome as part of the neutrophil NADPH oxidase may influence intracellular superoxide levels depending on local oxygen tension. Superoxide levels were otherwise shown to determine the EPO production in hepatoma cell lines. By double immunofluorescence labeling the alpha-subunit of cytochrome b558 (alpha-SU) and 5'NT were simultaneously localized in rat kidney and liver, and in the kidney Epo mRNA and alpha-SU were double-labeled. Positive signal for alpha-SU was found in the majority of renal peritubular fibroblasts in the cortex and outer medulla, and in Ito cells. In both organs, the cells that coexpress 5'NT and Epo mRNA also contain an immunoreactivity for alpha-SU. In these cells, cytochrome b558 as part of an NADPH oxidase may be involved in a presumptive oxygen sensing mechanism using H2O2 as a possible second messenger for EPO gene regulation.

5'-Nucleotidase↗

PubMed1997

Activation of ecto-5'-nucleotidase by protein kinase C and its role in ischaemic tolerance in the canine heart.

1. Ischaemic preconditioning (IP) protects the myocardium against irreversible ischaemic injury by activating protein kinase C (PKC). The mechanism by which PKC protects the myocardium is unknown. We have shown that PKC increases the activity of ecto-5'-nucleotidase (ecto-5'-N) and thereby the production of adenosine in cardiomyocytes which may protect the myocardium against ischaemia-reperfusion injury in vivo. 2. The objective of this study was to elucidate the possible role of PKC-induced activation of ecto-5'-N in the cardioprotection associated with IP in the canine heart. 3. IP increased the activities of both ecto-5'-N and PKC, and minimized ischaemic damage (infarct size: 7.5 +/- 1.8 vs. 42.3 +/- 2.8%, P < 0.01 vs. the control group). Treatment with the PKC activator (4 beta-phorbol 12-myristate-13-acetate) also reduced infarct size (13.5 +/- 2.9%, P < 0.01 vs. the control group). 8-Sulfophenyltheophylline (an antagonist of adenosine receptors) or alpha,beta-methyleneadenosine 5'-diphosphate (an inhibitor of ecto-5'-N) eliminated the cardioprotective effect of the PKC activator (infarct size: 36.6 +/- 3.9 and 34.7 +/- 4.2%, respectively), suggesting that PMA limits infarct size by increasing the activity of ecto-5'-N and the adenosine level. 4. The PMA-induced cardioprotection was blunted by GF109203X (an inhibitor of PKC, infarct size: 36.2 +/- 3.1%), but not by pretreatment with dexamethasone (infarct size, 14.2 +/- 2.6%). 5. We conclude that the PMA- and IP-induced cardioprotection is attributable to phosphorylation and activation of ecto-5'-N.

5'-Nucleotidase↗

PubMed1998

Analysis of the influence of nucleotidases on the apparent activity of exogenous ATP and ADP at P2Y1 receptors.

1. ADP is a potent agonist of rat and human P2Y1 purinoceptors. ATP is a weak competitive antagonist. This study analyses the situation in which P2Y1 receptors are exposed to ATP in the presence of exogenous ecto-nucleotidases (apyrases) that have high or low ATPase/ADPase activity ratio. 2. Rat brain capillary endothelial cells of the B10 clone express P2Y1 receptors that couple to intracellular Ca2+ mobilization. They have low endogenous ecto-ATPase and ecto-ADPase activities. 3. ATP did not raise intracellular Ca2+ in B10 cells. Addition of apyrases III or VII (1 u ml(-1)) to ATP treated cells induced large intracellular Ca2+ transients. Apyrases had no action in the absence of ATP. 4. A 1 u ml(-1) apyrase III solution generated 20 microM ADP from 0.1 mM ATP within 15 s. This concentration of ADP was sufficient to produce maximal activation of P2Y1 receptors. 5. ATP was a full agonist of P2Y1 receptors in the presence of 1 u ml(-1) apyrase III. Dose response curves for the apparent actions of ATP were bell shaped in the presence of 0.1 u ml(-1) apyrase III. Apyrase III did not alter ADP dose response curves when coincubated with ADP for 15 s. 6. Apyrase VII (1 u ml(-1)) shifted dose response curves for the actions of ADP to larger concentrations. It induced a bell shaped ATP dose response curve. 7. Results suggest that ATPDases prevent P2Y1 receptor activation by degrading ADP but may contribute to P2Y1 receptor activation by generating ADP from ATP.

Adenosine Diphosphate↗

PubMed2002

Real-time quantitative PCR assays for deoxycytidine kinase, deoxyguanosine kinase and 5'-nucleotidase mRNA measurement in cell lines and in patients with leukemia.

The relative levels of the deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK), and the 5'-nucleotidase (5'-NT) are of importance for the effect of many nucleoside analogues used in the treatment of hematological malignancies. To elucidate dCK, dGK and 5'-NT gene expressions in cell lines and in samples from patients with leukemia, we have established a real-time quantitative PCR (RQ-PCR) method. From the available dCK, dGK and 5'-NT cDNA sequences we designed specific primers and fluorogenic probes for the respective genes. The mRNA of dCK, dGK and 5'-NT was also measured by semi-quantitative RT-PCR, the enzyme activities by a radioactive substrate-based technique and Western blot was used to measure the amount of dCK and dGK protein. A MOLT-4 wild-type and its 9-beta-D-arabinofuranosylguanine (Ara-G)-resistant subline was used for the methods comparisons and the RQ-PCR assay was used in 35 samples from pediatric patients with ALL and AML. The results from RQ-PCR for the cell lines were in agreement with the semi-quantitative RT-PCR. The mRNA expression for dCK, dGK and 5'-NT (expressed as the ratio of the respective gene and the reference gene) in pediatric ALL and AML patients showed a large interindividual variability from 0.06 to 2.34, non-detectable to 0.06 and 0.04 to 0.30, respectively. These results show that the quantitative evaluation by RQ-PCR is a valuable tool in the determination of dCK, dGK and 5'-NT mRNA levels in cell lines and in clinical samples which were expressed at various levels. This rapid, convenient and specific method is suitable for further studies of these genes in clinical samples.

5'-Nucleotidase↗

PubMed1976

Subcellular distribution and activity in different rat tissues of a deoxyinosine-activated nucleotidase.

A nucleotidase (EC 3.1.3.31) isolated previously from rat liver cytosol was specifically measured in 14 different rat tissues and in subcellular fractions of liver and spleen, taking advantage of the stimulation exerted on it by deoxyinosine. The intracellular distribution studies showed that the enzyme is located almost entirely in the soluble cytoplasm except for the possible presence of 1-2% of the enzyme in the nucleus. The enzyme was present in various amounts in all the tissues studied. Spleen, thymus, and intestinal mucosa showed higher specific activities than any other tissue. On a per cell basis spleen, liver and intestinal mucosa had the highest enzyme activity, whereas bone marrow, brain, thymus, heart and skeletal muscle had activities in the lower range. The results may suggest that the enzyme plays a role in the recovery of endogenous nuclear material for nucleic acid synthesis.

Animals↗

PubMed2003

Biochemical and mass spectrometric characterization of soluble ecto-5'-nucleotidase from bull seminal plasma.

Ecto-5'-nucleotidase (ecto-5'-NT) is a glycosylphosphatidylinositol-anchored membrane-bound protein that is ubiquitous in mammalian tissues. It is a target for a number of therapeutic drugs since increased levels of the enzyme correlate with various disease states. In this investigation, we describe the properties of a soluble ecto-5'-NT derived from bull seminal plasma. The protein was highly heterogeneous as demonstrated by chromatofocusing and two-dimensional PAGE. Sequencing analyses revealed a truncated polypeptide lacking the glycosylphospatidylinositol attachment site, suggesting that it is produced post-translationally by cleavage at Gln(547) and/or Phe(548). Heterogeneity was largely due to differential glycosylation, especially in the oligosaccharides linked to Asn(403). Significant differences in substrate specificity were observed between isoforms and, on the basis of molecular-modelling studies, were interpreted in terms of variable glycosylation causing steric hindrance of the substrate-binding site. Thus the soluble forms of ecto-5'-NT found in bull seminal plasma are unique both biochemically and structurally, and have a putative role in signalling interactions with spermatozoa following ejaculation and capacitation in the female reproductive tract.

5'-Nucleotidase↗

PubMed1992

Purification and properties of 3'-nucleotidase of Leishmania donovani.

A surface membrane 3'-nucleotidase from Leishmania donovani promastigotes has been purified to SDS/PAGE homogeneity. The enzyme has apparent subunit molecular mass of 38 kDa, pI 5.8 and a broad pH optimum, 5.5-7.5. EDTA partially inhibited the enzyme activity, which was fully restored by Co2+; Mg2+, Ca2+ or Mn2+ had no effect on the activity. ZnCl2 or dithiothreitol at 1 mM was inhibitory at pH 7.5, but was without effect at pH 5.5, whereas at both pH values 5 mM of either compound inhibited the enzyme. The substrate-specificity of the purified enzyme is restricted to ribonucleoside 3'-phosphates. 3'-AMP and 3'-IMP are the best substrates, whereas ADP, ATP, 2'-deoxyadenosine 3'-phosphate and 5'-AMP are competitive inhibitors of the enzyme. The enzyme showed low latency in intact-cell preparations. The kinetic properties and the surface membrane localization of the enzyme suggest its implication in the formation of nucleosides from 3'-nucleotides of the parasite's host.

Adenine Nucleotides↗

PubMed1994

Purification and some properties of an IMP-specific 5'-nucleotidase from yeast.

An IMP-hydrolysing enzyme was purified to homogeneity from yeast extract. It was a soluble protein with an apparent molecular mass of 220 kDa, with a subunit molecular mass of 55 kDa. It was highly specific for IMP, and there was virtually no detectable activity with the other purine and pyrimidine nucleotides tested, including AMP and dIMP. The enzyme had a pH optimum of 6.0-6.5. Its activity was absolutely dependent on bivalent metal salts: Mg2+ was most potent, followed by Co2+ and Mn2+. The velocity/substrate-concentration plot of the enzyme was slightly sigmoidal (h = 1.7) and the s0.5 was 0.4 mM. ATP stimulated the enzyme by decreasing both h and s0.5. Diadenosine tetraphosphate stimulated the enzyme as effectively as ATP. Although the properties of the enzyme are similar to those of the IMP/GMP 5'-nucleotidase identified in various animals [Itoh (1993) Comp. Biochem. Physiol. 105B, 13-19], the substrate specificity of the former was much more strict than the latter.

5'-Nucleotidase↗

PubMed1994

Homogeneous pyrimidine nucleotidase from human erythrocytes: enzymic and molecular properties.

A pyrimidine nucleotidase with unique specificity has been obtained for the first time as an homogeneous protein from the cytosolic fraction of human erythrocytes. Both conventional chromatography and f.p.l.c. techniques have been used in the purification procedure. The final enzyme preparation gave a single protein band of M(r) = 23,500 on SDS/PAGE under both reducing and non-reducing conditions. The native enzyme was eluted at M(r) = 45,000 in gel filtration chromatography on Superose 12, suggesting a dimeric structure. Amino acid analysis was consistent with an acidic isoelectric point and revealed the presence of six half-cystine and two methionine residues per subunit. The enzyme was active on a variety of pyrimidine nucleoside monophosphates, being most active on the 3'-monophosphates. Km values for 3'dUMP, 3'UMP, 5'dUMP, 5'UMP, 5-fluoro-2'dUMP, ranged from 192 microM to 1.15 mM. The enzyme activity was inhibited by both reaction products, orthophosphate, and the nucleoside formed. Product inhibition studies suggested an Ordered Uni-Bi mechanism for the reaction. The enzyme required Mg2+ for its activity, while heavy metals cations were strongly inhibitory. The enzyme activity was inhibited by metal chelating agents and it was sensitive to thioreactive reagents. The isoelectric point was 5.4 and the optimum activity pH was 6.5.

5'-Nucleotidase↗

PubMed1997

Differentiation of BCS-TC2 human colon adenocarcinoma cells by sodium butyrate: increase in 5'-nucleotidase activity.

We have analysed the major effects of sodium butyrate on the morphology, protein content and induction of epithelial differentiation markers in human colon adenocarcinoma BCS-TC2 cells. Sodium butyrate alters the cell morphology, inducing a larger cellular size, flattening and vacuolization. The protein content per cell increases, whereas the proliferation rate is reduced. Moreover, cell death by apoptosis is also observed. Butyrate-treated cells show higher levels of alkaline phosphatase activity and carcinoembryonic antigen, suggesting that this agent induces the in vitro differentiation of BCS-TC2 cells. These effects are reversible and time and dose dependent. In addition, we have observed that the ectoenzyme 5'-nucleotidase activity also increases during this treatment, suggesting that it could be considered as a new differentiation marker for this type of carcinoma cells. These results contribute to the understanding of the action of sodium butyrate as a potential cancer chemotherapeutic agent.

5'-Nucleotidase↗

PubMed2000

Ultracytochemical localization of 5'-nucleotidase activity in human ejaculated spermatozoa.

5'-nucleotidase, an adenosine producing enzyme with a glycosylphosphatidylinositol-anchored structure, was localized in human ejaculated spermatozoa. The poly-L-lysine-coated dish method was used to prepare the specimens, and the cerium method was employed for electron-microscopic enzyme localization. Precipitates indicating enzyme activity were detected on the outer side of the external plasma membrane of the acrosomal region. This enzyme may play a role in sperm motility and male infertility.

5'-Nucleotidase↗

PubMed1996

Decreased number of CD73 (ecto-5'-nucleotidase) molecules on lymphocytes from patients with primary immunoglobulin deficiencies. Correlation between number of CD73 molecules and T-lymphocyte function in vitro.

CD73 is a bifunctional glycosyl phosphatidylinositol anchored leucocyte differentiation antigen which has specific ecto-5'-nucleotidase (ecto-5'-NT) activity and is an accessory T-lymphocyte activation molecule. The aim of the present study was to investigate the CD73 expression on blood mononuclear cells (BMC) from a group of patients with primary immunoglobulin deficiency (IGD). This group of patients had both significantly decreased levels of ecto-5'-NT on BMC (P = 0.002) and decreased numbers of CD73 molecules per CD73+ lymphocyte (P = 0.01). Five of the 10 patients had a decreased percentage of CD73+ lymphocytes. Among B-lymphocytes the patients had normal percentages of CD73+ cells but four of the 10 patients had numbers of CD73 molecules per CD73+ B-lymphocyte below the normal range. Among CD4-lymphocytes three out of 10 patients had percentages of CD73+ below the normal range and four out of 10 patients had decreased percentages of CD73+ CD8-lymphocytes. Significant correlations were found between in vitro proliferative responses to mitogens and the number of CD73 molecules per CD73+ lymphocyte (rs = 0.60, P < 0.01) and per CD73+ CD8-lymphocyte (rs = 0.64, P < 0.02). In addition, a positive correlation was found between ability to proliferate and level of ecto-5'-NT on BMC (rs = 0.53, P < 0.05). Furthermore the ability of BMC to synthesize ecto-5'-NT was studied. During 2 days culture ecto-5'-NT activity increased markedly on BMC from both patients and healthy donors. The level of activity on BMC from all patients attained levels higher than on freshly isolated BMC from healthy donors. This shows that the decreased levels of ecto-5'-NT found on freshly isolated BMC from patients with IGD is due to defective regulation of the enzyme activity in vivo.

5'-Nucleotidase↗

PubMed2000

Chronic non-spherocytic haemolytic anaemia due to congenital pyrimidine 5' nucleotidase deficiency: 25 years later.

In 1972, Valentine et al described, under the name of 'non-spherocytic haemolytic anaemia, high red cell ATP and ribose phosphate pyrophosphokinase (RPK; EC 2.7.6.1) deficiency', an obscure congenital haemolytic anaemia with the characteristic feature of red blood cell basophilic stippling. The activity of Embden-Meyerhof pathway and hexose monophosphate shunt were normal, and the concentrations of reduced glutathione and of ATP were raised 2 SD above the normal mean. The low values of RPK also encountered were considered to be an epiphenomenon rather than a causative defect. One year later, further studies performed in two new kindreds with the same haemolytic disorder associated with persistent basophilic stippling were described under the name of 'haemolytic disorders associated with increased ATP'. In 1974, two new and important observations contributed to the final identification of the disease: the patients' red blood cells (RBCs) contained large amounts of nucleotides (pyrimidine nucleotides), and in all cases they were deficient in a hitherto unrecognized enzyme called pyrimidine 5' nucleotidase (P5N). In conclusion, all these cases were formerly referred to as 'high ATP syndromes' because of the erroneous assumption that the large number of nucleotides within deficient RBCs were adenine phosphate rather than pyrimidine phosphate. Twenty-five years after its description, P5N deficiency has been reported in about 35 unrelated families from different parts of the world, and it has become one commonly identified cause of hereditary non-spherocytic haemolytic anaemia due to RBC enzymopathy. Genetic transmission is via the autosomic recessive mode, and only homozygous or compound heterozygous are clinically affected. Family members who are biochemically heterozygous are haematologically normal and difficult to detect. Unfortunately, the precise gene mutation or mutations causing the disease remain unknown.

5'-Nucleotidase↗

PubMed1993

[Pyrimidine 5'-nucleotidase deficiency as the congenital cause of nonspherocytic hemolytic anemia].

An 11-year-old girl had marked haemolytic anaemia since the first year of life. Physical examination revealed scleral and cutaneous icterus and slight splenomegaly. Haemoglobin concentration was reduced to 9.5 g/dl, while platelet count and bilirubin concentration were increased (350,000/microliter and 2.2 mg/dl, respectively). The erythrocytes showed marked basophilic stippling, its extent typical of pyrimidine-5'-nucleotidase deficiency. The enzyme activity in the erythrocytes was 15% of normal. Deficiency of this enzyme, inherited as an autosomal recessive, is probably one of the most common erythrocyte enzyme deficiencies causing haemolytic anaemia. It brings about the intracellular accumulation of pyrimidine nucleotides which via secondary metabolic changes causes an accelerated destruction of erythrocytes. There is no known causative treatment: splenectomy is ineffective against the anaemia.

5'-Nucleotidase↗

PubMed1995

The salivary gland-specific apyrase of the mosquito Aedes aegypti is a member of the 5'-nucleotidase family.

The saliva of hematophagous insects contains a variety of pharmacologically active substances that counteract the normal hemostatic response to injury in vertebrate hosts. The yellow-fever mosquito, Aedes aegypti, secretes an apyrase that inhibits ADP-dependent platelet aggregation. Apyrase was purified as an active enzyme from adult female salivary glands and subjected to tryptic digestion, and the resulting peptides were sequenced. The amino acid sequences obtained match the conceptual translation product of a cDNA clone isolated from an adult female salivary gland library. Sequence comparisons indicate similarities with a ubiquitous family of 5'-nucleotidases. The mosquito protein differs from other members of the family by lacking a carboxyl-terminal hydrophobic domain. The apparent conversion of a gene encoding an enzyme involved in a common metabolic event at the cellular level to a gene involved in the antihemostatic response of mosquitoes illustrates one way this particular insect has adapted to the challenges of bloodfeeding.

5'-Nucleotidase↗

PubMed1996

Nucleotidase activities of the 26 S proteasome and its regulatory complex.

The 26 S proteasome can be assembled from the multicatalytic protease (or 20 S proteasome) and a large multisubunit regulatory complex in an ATP-dependent reaction. The 26 S proteasome and its regulatory complex were purified from rabbit reticulocytes for characterization of their nucleotidase properties. Both particles hydrolyze ATP, CTP, GTP, and UTP to the corresponding nucleoside diphosphate and inorganic phosphate. The Km values for hydrolysis of specific nucleotides by the 26 S proteasome are 15 microM for ATP and CTP, 50 microM for GTP, and 100 microM for UTP; Km values for nucleotide hydrolysis by the regulatory complex are 2-4-fold higher for each nucleotide. Several ATPase inhibitors (erythro-9-[3-(2-hydroxynonyl)]adenine, oligomycin, ouabain, and thapsigargin) had no effect on ATP hydrolysis by either complex whereas known inhibitors of proteolysis by the 26 S enzyme (hemin, N-ethylmaleimide (NEM), and vanadate) significantly reduced ATP hydrolysis by both particles. Hydrolysis of all nucleotides was inhibited by 5 mM NEM but only GTP and UTP hydrolysis was significantly reduced at 50 microM NEM. The 15 microM Km for ATP hydrolysis by the 26 S proteasome is virtually identical to the observed Km of 12 microM ATP for Ub-conjugate degradation. Although nucleotide hydrolysis is required for protein degradation by the 26 S proteasome, nucleotide hydrolysis and peptide bond cleavage are not strictly coupled. Substrate specificity constants (kcat/Km) are similar for hydrolysis of each nucleotide, yet GTP and UTP barely supported Ub-conjugate degradation. Further evidence that nucleotide hydrolysis is not coupled to peptide bond cleavage was obtained using N-acetyl-leucyl-leucyl-norleucinal (LLnL). This compound inhibited peptide hydrolysis by the multicatalytic protease and Ub-conjugate degradation by the 26 S proteasome, but it had little effect on ATP or UTP hydrolysis by the 26 S enzyme.

Adenosine Triphosphatases↗

PubMed1996

Inhibition of ecto-5'-nucleotidase by nitric oxide donors. Implications in renal epithelial cells.

We evaluated, in renal epithelial cells with a proximal tubule phenotype, the effect of nitric oxide (NO) on ecto-5 -nucleotidase (5'-N U), the underlying mechanism and its functional consequence. Sodium nitroprusside (SNP, 1-1000 microM), a NO donor, inhibited 5'-NU activity in a time- and concentration-dependent manner. Consequently, NO blunted the inhibition by extracellular cyclic AMP (cAMP, 10-1000 microM) of sodium-phosphate cotransport, a pathway which involves degradation of adenosine monophosphate (AMP) by 5'-NU. SNP-induced inhibition of 5'-NU was not mediated by cyclic GMP, since it was not mimicked by atrial natriuretic peptide, and was reproduced by isosorbide dinitrate and sodium nitrate, two NO donors. SNP and genuine NO decreased the activity of 5'-NU in renal homogenates, and the effect of SNP was potentiated by dithiothreitol and glutathione, but not by nicotinamide adenine dinucleotide. In vivo in rats, kidney ischemia/reperfusion, which activates inducible NO-synthase, inhibited renal 5'-NU. This inhibition was prevented by Nomega-nitro-L-arginine methyl ester, a NO-synthase inhibitor. These results indicate that: (i) NO-related activity inhibited the activity of an ecto-enzyme, 5'-NU, most likely through S-nitrosylation of the enzyme; (ii) inhibition of 5'-NU activity by NOx, which can occur in vivo under pathophysiological conditions, affected the extent to which extracellular cAMP inhibited sodium-Pi cotransport.

5'-Nucleotidase↗