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1.
Arch Biochem Biophys ; 364(2): 235-40, 1999 Apr 15.
Article in English | MEDLINE | ID: mdl-10190979

ABSTRACT

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.


Subject(s)
5'-Nucleotidase/metabolism , Adenosine Monophosphate/metabolism , Myocardium/enzymology , Thymine Nucleotides/pharmacology , Adenosine Triphosphate/metabolism , Animals , Enzyme Inhibitors/pharmacology , Hydrolysis , Male , Rats , Rats, Sprague-Dawley
2.
Antimicrob Agents Chemother ; 38(6): 1230-8, 1994 Jun.
Article in English | MEDLINE | ID: mdl-8092819

ABSTRACT

The (-) and (+) enantiomers of the nucleoside analog cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (2',3'-dideoxy-5-fluoro-3'-thiacytidine; FTC) have been shown to inhibit hepatitis B virus replication in vitro in HepG2 derivative 2.2.15 (subclone P5A) cells. (-)-FTC and (+)-FTC were anabolized to 5'-monophosphate, 5'-diphosphate, and 5'-triphosphate in this cell line. (-)-FTC was more efficiently phosphorylated to the 5'-triphosphate than (+)-FTC, and levels of 3.6 and 0.2 pmol/10(6) cells, respectively, were detected after incubation with 1 microM compound for 24 h. A time course study showed that nucleotides were formed rapidly in a dose-dependent manner and reached a steady-state intracellular concentration by 3 to 6 h. The intracellular half-life of (-)-FTC 5'-triphosphate was 2.4 h. Both (-)- and (+)-FTC were converted to diphosphocholine derivatives, analogous to CDP-choline, but only (+)-FTC was converted to the diphosphoethanolamine derivative, analogous to CDP-ethanolamine. (-)-FTC was not detectably deaminated at either the nucleoside or nucleotide level. (+)-FTC was partially deaminated by these cells. The transport of (-)-and (+)-FTC was examined in HepG2 cells. (+)-FTC enters these cells by way of the nitrobenzylthioinosine-susceptible, equilibrative nucleoside transporter. In contrast, the influx of (-)-FTC was only partially susceptible to inhibitors of nucleoside transport, indicating that (-)-FTC may have multiple transport mechanisms. These metabolic results are consistent with the conclusion that (-)-FTC 5'-triphosphate mediates the anti-hepatitis B virus activity of (-)-FTC.


Subject(s)
Antiviral Agents/metabolism , Zalcitabine/analogs & derivatives , Cell Line , Dose-Response Relationship, Drug , Emtricitabine/analogs & derivatives , Half-Life , Humans , Liver/metabolism , Stereoisomerism , Zalcitabine/metabolism
3.
Antimicrob Agents Chemother ; 36(2): 283-6, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1318676

ABSTRACT

The transport of the anti-varicella-zoster virus agent 6-methoxypurine arabinoside and its 2'-O-valerate prodrug, 170U88, was investigated by using the human erythrocyte model. The influx of 6-methoxypurine arabinoside was found to occur primarily by means of the nucleoside transporter. (i) Influx was nonconcentrative and saturable (Km = 106 +/- 2 microM). (ii) The inhibitors of nucleoside transport, nitrobenzylthionosine, dipyridamole, and dilazep, inhibited the influx of 10 microM 6-methoxypurine arabinoside by greater than 94%. (iii) Influx was inhibited by nucleosides but not by nucleobases. (iv) 6-Methoxypurine arabinoside was a competitive inhibitor (Ki = 129 +/- 10 microM) of adenosine influx, and adenosine (Km = 160 +/- 9 microM) was found to be a competitive inhibitor (Ki = 134 +/- 9 microM) of 6-methoxypurine arabinoside influx. By contrast, the influx of 170U88 occurred by means of nonfacilitated diffusion. (i) Influx was linearly dependent on the 170U88 concentration. (ii) Influx was not inhibited by nucleobases, nucleosides, or inhibitors of nucleoside transport.


Subject(s)
Antiviral Agents/pharmacokinetics , Arabinonucleosides/pharmacokinetics , Erythrocytes/metabolism , Herpesvirus 3, Human/drug effects , Prodrugs , Antiviral Agents/pharmacology , Arabinonucleosides/chemistry , Arabinonucleosides/pharmacology , Chemical Phenomena , Chemistry, Physical , Humans , In Vitro Techniques
4.
Cancer Res ; 50(6): 1817-21, 1990 Mar 15.
Article in English | MEDLINE | ID: mdl-2306735

ABSTRACT

Nitrobenzylthioinosine (NBMPR), dipyridamole, and dilazep, potent inhibitors of nucleoside transport, were found to be ineffective in preventing 9-beta-D-arabinofuranosylguanine (ara-G)-induced inhibition of MOLT 4 and CCRF CEM cell growth, ara-G (2.0 microM) was metabolized to 9-beta-D-arabinofuranosylguanine 5'-triphosphate in MOLT 4 cells, and the levels of this metabolite were not affected by the presence of 5.0 microM NBMPR in the incubation medium. Permeation of the MOLT 4 cell membrane by ara-G occurred primarily by means of the NBMPR-sensitive nucleoside transport system. However, a residual transport component accounting for 10-20% of the total transport activity was demonstrated in the presence of NBMPR. This component was inhibited by adenine and hypoxanthine but not by dilazep, dipyridamole, or other nucleosides. In contrast, inhibitors of nucleoside transport readily reversed the cytotoxic effect of 7-deazaadenosine (tubercidin) in both MOLT 4 and CCRF CEM cells. The levels of tubercidin 5'-triphosphate formed from 2.0 microM tubercidin in MOLT 4 cells were reduced by 80% in the presence of 5.0 microM NBMPR. The influx of tubercidin into MOLT 4 cells was found to occur primarily by means of the NBMPR-sensitive nucleoside transport system. This same system mediated the transport of ara-G into human erythrocytes.


Subject(s)
Arabinonucleosides/metabolism , Erythrocytes/metabolism , Inosine/analogs & derivatives , Thioinosine/analogs & derivatives , Tumor Cells, Cultured/metabolism , Arabinonucleosides/blood , Biological Transport/drug effects , Cell Division/drug effects , Cell Line , Dilazep/pharmacology , Dipyridamole/pharmacology , Erythrocytes/drug effects , Humans , Kinetics , Leukemia-Lymphoma, Adult T-Cell , Purines/pharmacology , Ribonucleosides/pharmacology , Thioinosine/pharmacology , Tubercidin/metabolism , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/drug effects
5.
Biochem Pharmacol ; 38(3): 509-17, 1989 Feb 01.
Article in English | MEDLINE | ID: mdl-2537081

ABSTRACT

Evidence has been obtained for the metabolic formation of small amounts (1-2% of the ATP pool) of 3-deazaadenosine 5'-triphosphate (c3ATP) from 3-deazaadenosine (c3Ado) in mouse cytolytic lymphocytes and mouse resident peritoneal macrophages. With intact leukocytes, pharmacological evidence was obtained that adenosine kinase was not the enzyme chiefly responsible for the phosphorylation of c3Ado. Moreover, in the presence of MgCl2, NaCl and IMP, purified rat liver 5'-nucleotidase catalyzed the phosphorylation of c3Ado to 3-deazaadenosine 5'-monophosphate (c3AMP). Two lines of evidence suggest that the metabolic formation of c3ATP is not involved in the inhibition of leukocyte function caused by c3Ado. First, the inhibitory action of c3Ado on antibody-dependent phagocytosis and lymphocyte-mediated cytolysis was reversed markedly upon removal of the drug from the medium. However, the intracellular content of c3ATP remained constant in lymphocytes and macrophages after removal of c3Ado. Second, in macrophages and in lymphocytes, similar intracellular amounts of c3ATP were formed from both c3Ado and 3-deazaadenine under conditions in which the former was biologically active and the latter was essentially inactive. Thus, it appears unlikely that the novel c3ATP metabolite is of relevance for the mechanism of action of c3Ado in mouse leukocytes.


Subject(s)
Adenosine Triphosphate/metabolism , Anti-Bacterial Agents/metabolism , Lymphocytes/metabolism , Macrophages/metabolism , Tubercidin/metabolism , 5'-Nucleotidase , Aminoglycosides , Animals , In Vitro Techniques , Lymphocytes/drug effects , Macrophages/drug effects , Mice , Nucleotidases/pharmacology , Phosphorylation , Tubercidin/pharmacology
7.
J Biol Chem ; 262(12): 5748-54, 1987 Apr 25.
Article in English | MEDLINE | ID: mdl-3471758

ABSTRACT

The demonstrated in vitro and in vivo activity of 3'-azido-3'-deoxythymidine (N3dThd) against the infectivity and the cytopathic effect of human immunodeficiency virus has prompted an investigation of the mechanism by which this nucleoside analogue permeates the cell membrane. As with the transport of thymidine, the influx of N3dThd into human erythrocytes and lymphocytes was nonconcentrative during short incubation times (less than 5 min) which did not allow significant metabolism of this nucleoside. However, in contrast with thymidine transport, the initial velocity of N3dThd influx was strictly a linear function of nucleoside concentration (0.5-10 mM), without evidence of saturability; insensitive to micromolar concentrations of potent inhibitors of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep); insensitive to a 1000-fold excess of other nucleosides (thymidine, uridine, 2-chloroadenosine); and relatively insensitive to temperature, with Q10 values (37-27 degrees C) of 1.4 and 2.7 for N3dThd and thymidine, respectively, determined in erythrocytes. Although the above results indicate that N3dThd permeates the cell membrane chiefly by nonfacilitated diffusion and not via the nucleoside transporter, millimolar concentrations of this nucleoside analogue were observed to inhibit both zero-trans influx of thymidine and efflux of thymidine from [3H]thymidine-loaded erythrocytes. The partition coefficients (1-octanol:0.1 M sodium phosphate, pH 7.0) of N3dThd and thymidine were determined to be 1.26 and 0.064, respectively. The unusual ability of N3dThd to diffuse across cell membranes independently of the nucleoside transport system may be attributed to the considerable lipophilicity imparted to this molecule by the replacement of the 3'-hydroxyl group of thymidine with an azido moiety.


Subject(s)
Antiviral Agents/blood , Erythrocytes/metabolism , Lymphocytes/metabolism , Thymidine/analogs & derivatives , Biological Transport , Cell Membrane Permeability , Diffusion , Humans , Kinetics , Thymidine/blood , Thymidine/pharmacology , Zidovudine
8.
Proc Natl Acad Sci U S A ; 82(12): 4060-4, 1985 Jun.
Article in English | MEDLINE | ID: mdl-3858863

ABSTRACT

3-Deazaadenosine (c3Ado) has been reported to inhibit a number of cellular functions. These biological effects of c3Ado have generally been attributed to its ability to act as inhibitor and substrate of S-adenosylhomocysteine hydrolase. In this report, it is revealed by fluorescence microscopy that c3Ado caused disorganization of the microfilament system of mouse macrophages at concentrations (greater than or equal to 5 microM) similar to those that inhibited antibody-dependent phagocytosis and zymosan-stimulated H2O2 production by these cells. Inhibition of phagocytosis and perturbation of microfilaments by c3Ado were completely abrogated by washing the macrophages free of this agent and allowing the cells a 30-min recovery period. Furthermore, these effects of c3Ado on phagocytosis and microfilaments appeared to be independent of the increase in S-adenosylhomocysteine and S-3-deazaadenosylhomocysteine that occurred in these macrophages. First, periodate-oxidized adenosine and 3-deaza(+/-)aristeromycin, two other inhibitors of S-adenosylhomocysteine hydrolase that caused greater increases in macrophage S-adenosylhomocysteine than did c3Ado, had no effect on either phagocytosis or microfilaments. Second, pretreatment of macrophages with periodate-oxidized adenosine (to inhibit S-adenosylhomocysteine hydrolase) prevented the subsequent metabolism of c3Ado to S-3-deazaadenosylhomocysteine but did not diminish the effects of c3Ado on phagocytosis or microfilaments. These results demonstrate that c3Ado can perturb the microfilament system of cells and provide an alternative mechanism for the biological effects of c3Ado.


Subject(s)
Cytoskeleton/drug effects , Macrophages/drug effects , Ribonucleosides/pharmacology , Tubercidin/pharmacology , Actins/metabolism , Adenosine Triphosphate/metabolism , Animals , Macrophages/ultrastructure , Male , Mice , Peroxides/metabolism , Phagocytosis/drug effects , S-Adenosylhomocysteine/metabolism
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