Purine and pyrimidine transport and phosphoribosylation and their interaction in overall uptake by cultured mammalian cells. A re-evaluation.

The zero-trans uptake of purines and pyrimidines was measured in suspensions of Novikoff rat hepatoma, mouse L, P388 mouse leukemia, and Chinese hamster ovary cells by a rapid kinetic technique which allows the determination of uptake time points in intervals as short as 1.5 s. Kinetic parameters for purine/pyrimidine transport were determined by measuring substrate influx into cells in which substrate conversion to nucleotides was negligible either due to lack of the appropriate enzymes or to depletion of the cells of ATP (5'-phosphoribosylpyrophosphate), and by computer fitting exact, integrated rate equations derived for various carrier-mediated transport models directly to zero-trans influx data. The results indicate that different carriers function in the transport of hypoxanthine/guanine, adenine, and uracil with substrate:carrier association constants (K) at 24 degrees C of 300 to 400 muM, 2 to 3 mM, and about 14 mM, respectively, for Novikoff cells. K and Vmax for hypoxanthine transport by L and P388 cells are similar to those for Novikoff cells, but the transport capacity of Chinese hamster ovary cells is much lower and K = 1500 muM. All transport systems are completely symmetrical. Hypoxanthine transport is so rapid that an intracellular concentration of free hypoxanthine (90%) close to that in the medium is attained within 20 to 50 s of incubation at 24 degrees C, at least at extracellular concentrations below K. In cells in which conversion to nucleotides is not blocked free hypoxanthine accumulates intracellularly to steady state levels with equal rapidity and thereafter the rate of hypoxanthine uptake into total cell material is strictly a function of the rate of phosphoribosylation. The low Km systems for hypoxanthine (1 to 9 muM) and adenine (0.2 to 40 muM) uptake detected previously in many types of cells reflect the substrate saturation of the respective phosphoribosyltransferases rather than of the transport system.     

Thymidine transport by cultured Novikoff hepatoma cells and uptake by simple diffusion and relationship to incorporation into deoxyribonucleic acid

The initial rate of thymidine-³H incorporation into the acid-soluble pool by cultured Novikoff rat hepatoma cells was investigated as a function of the thymidine concentration in the medium. Below, but not above 2 µM, thymidine incorporation followed normal Michaelis-Menten kinetics at 22°, 27°, 32°, and 37°C with an apparent K_m of 0.5 µM, and the V_max values increased with an average Q₁₀ of 1.8 with an increase in temperature. The intracellular acid-soluble ³H was associated solely with thymine nucleotides (mainly deoxythymidine triphosphate [dTTP]). Between 2 and 200 µM, on the other hand, the initial rate of thymidine incorporation increased linearly with an increase in thymidine concentration in the medium and was about the same at all four temperatures. Pretreatment of the cells with 40 or 100 µM p-chloromercuribenzoate for 15 min or heat-shock (49.5°C, 5 min) markedly reduced the saturable component of uptake without affecting the unsaturable component or the phosphorylation of thymidine. The effect of p-chloromercuribenzoate was readily reversed by incubating the cells in the presence of dithiothreitol. Persantin and uridine competitively inhibited thymidine incorporation into the acid-soluble pool without inhibiting thymidine phosphorylation. At concentrations below 2 µM, thymidine incorporation into DNA also followed normal Michaelis-Menten kinetics and was inhibited in an apparently competitive manner by Persantin and uridine. The apparent K_m and K_i values were about the same as those for thymidine incorporation into the nucleotide pool. The over-all results indicate that uptake is the rate-limiting step in the incorporation of thymidine into the nucleotide pool as well as into DNA. The cells possess an excess of thymidine kinase, and thymidine is phosphorylated as rapidly as it enters the cells and is thereby trapped. At low concentrations, thymidine is taken up mainly by a transport reaction, whereas at concentrations above 2 µM simple diffusion becomes the principal mode of uptake. Evidence is presented that indicates that uridine and thymidine are transported by different systems. Upon inhibition of DNA synthesis, net thymidine incorporation into the acid-soluble pool ceased rapidly. Results from pulse-chase experiments indicate that a rapid turnover of dTTP to thymidine may be involved in limiting the level of thymine nucleotides in the cell.     

Purine and pyrimidine transport in Schistosoma mansoni.

The uptake of purines and pyrimidines by adults of Schistosoma mansoni was studied. Cytosine, thymine, and uracil entered the worms entirely by diffusion. Adenine, guanine, hypoxanthine, and the nucleosides adenosine and uridine were absorbed in part by mediated systems. The results of inhibitor studies suggest the presence of 5 distinct transport sites for these latter compounds. The interaction of adenosine monophosphate with these sites was also studied.     

Purine and pyrimidine metabolism in human muscle and cultured muscle cells

Using radiochemical methods, we determined the activities of various enzymes of purine and pyrimidine metabolism in homogenates of human skeletal muscle and of cultured human muscle cells. Results show a large discrepancy between the enzyme activities in muscle and cultured cells. With regard to purine metabolism, adenylate (AMP) deaminase activity was only 1-3% in cultured cells compared to that in muscle, whereas the activity of adenosine deaminase, purine-nucleoside phosphorylase, adenosine kinase, adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase was 7-15-fold higher in the cultured cells. The enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase, orotidine 5'-monophosphate decarboxylase and uridine kinase showed activity of 100-200-fold higher in cultured cells than in adult muscle. The differences in enzyme activity are probably related to the low differentiation stage and the absence of contractile activity in the cultured muscle cells. Care must be taken when using these cells as a model for studying purine and pyrimidine metabolism of adult myofibers.     

Cytochalasin B. VI. Competitive inhibition of nucleoside transport by cultured Novikoff rat hepatoma cells

Cytochalasin B competitively inhibits the transport of uridine and thymidine by Novikoff rat hepatoma cells growing in suspension culture with apparent K_i''s of 2 and 6 µM, respectively, but has no effect on the intracellular phosphorylation of the nucleosides. Choline transport is not affected by cytochalasin B. Results from pulse-chase experiments indicate that cytochalasin B has no direct effect on the synthesis of RNA, DNA, or uridine diphosphate-sugars. The inhibition of uridine and thymidine incorporation into nucleic acids by cytochalasin B is solely the consequence of the inhibition of nucleoside transport.     

The deoxyribonucleoside transport systems of cultured Novikoff rat hepatoma cells

The transport of various deoxyribonucleosides by cultured Novikoff rat hepatoma cells (subline N1S1-67) follows normal Michaelis-Menten kinetics. The transport reactions are competitively inhibited by most heterologous deoxy- and ribonucleosides and by Persantin and Cytochalasin B. Comparisons of the transport kinetics of the various deoxyribonucleosides (K_m and V_max ) and of the K_m/K_i ratios for the inhibitions indicate that deoxythymidine, deoxyuridine and 5-fluordeoxyuridine are transported by a single system, whereas deoxycytidine and the purine deoxyribonucleosides are transported by other systems. The data suggest that deoxyadenosine, deoxyguanosine and deoxyinosine, are not transported by a single system, but the number of transport systems involved could not be established unequivocally. Similar comparisons also suggest that the deoxyribonucleosides are transported by different systems than the ribonucleosides. All deoxyribonucleoside transport systems are inhibited to about the same extent by Persantin (K_i = 1–2 μM) and Cytochalasin B (K_i = 4–12 μM). The inhibitions of deoxynucleoside transport resulted in corresponding apparent competitive inhibitions of their incorporation into nucleic acids.     

Purine and pyrimidine transport and permeation in human erythrocytes

Time courses of the uptake of radiolabeled hypoxanthine, adenine and uracil were measured by rapid kinetic techniques over substrate ranges from 0.02 to 5000 microM in suspensions of human erythrocytes at 25 or 30 degrees C. At concentrations above 25 microM, the rate of intracellular phosphoribosylation of hypoxanthine and adenine was insignificant relative to their rates of entry into the cell and time courses of transmembrane equilibration of the substrates could be measured and analyzed by integrated rate analysis. Hypoxanthine and uracil are transported by simple facilitated carriers with directional symmetry, high capacity and Michaelis-Menten constants of about 0.2 and 5 mM, respectively. Adenine is probably transported by a carrier with similar properties but no saturability was detectable up to a concentration of 5 mM. Cytosine entered the cells much more slowly than the other three nucleobases, and its entry seems not to be mediated by a carrier. The hypoxanthine transporter resembles that of one group of mammalian cell lines, which does not exhibit any overlap with the nucleoside transporter and is resistant to inhibitors of nucleoside transport. Results from studies on the effects of the nucleobases on the influx and countertransport of each other were complex and did not allow unequivocal conclusions as to the number of independent carriers involved. At concentrations below 5 microM, radiolabel from adenine and hypoxanthine accumulated intracellularly to higher than equilibrium levels. Part of this accumulation reflected metabolic trapping, especially when the medium contained 50 mM phosphate. But part was due to an apparent concentrative accumulation of free adenine and hypoxanthine up to 3-fold at medium concentrations much less than 1 microM and when cells were incubated in phosphate-free medium. This concentrative accumulation could be due to the functioning of additional high-affinity, low-capacity, active transport systems for adenine and hypoxanthine, but other factors could be responsible, such as saturable binding to intracellular components.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

Purine and pyrimidine transport in pathogenic protozoa: from biology to therapy

Purine salvage is an essential function for all obligate parasitic protozoa studied to date and most are also capable of efficient uptake of preformed pyrimidines. Much progress has been made in the identification and characterisation of protozoan purine and pyrimidine transporters. While the genes encoding protozoan or metazoan pyrimidine transporters have yet to be identified, numerous purine transporters have now been cloned. All protozoan purine transporter-encoding genes characterised to date have been of the Equilibrative Nucleoside Transporter family conserved in a great variety of eukaryote organisms. However, these protozoan transporters have been shown to be sufficiently different from mammalian transporters to mediate selective uptake of therapeutic agents. Recent studies are increasingly addressing the structure and substrate recognition mechanisms of these vital transport proteins.     

Purine and pyrimidine inhibitors of arginase.

1. Adenosine, inosine, adenine and uric acid are competitive inhibitors and cytidine and cytosine noncompetitive inhibitors of bovine liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1). 2. The affinity of the enzyme for these inhibitors was 10--100 times as great as for substrate in terms of Ki versus Km. 3. These nucleic acid metabolites may thus function in vivo to regulate the urea cycle. 4. Several naturally occuring competitive and noncompetitive inhibitors of arginase of unknown structure have been isolated from plant and animal tissue. From their properties and methods of isolation, they may be the purines and pyrimidines herein described. 5. These purines and pyrimidines have no effect on tryptic hydrolysis.     

Uridine transport in Novikoff rat hepatoma cells and other cell lines and its relationship to uridine phosphorylation and phosphorolysis.

Time courses of [³H]uridine uptake as a function of uridine concentration were determined at 25° in untreated and ATP-depleted wild-type and uridine kinase-deficient Novikoff cells and in mouse L and P388 cells, Chinese hamster ovary cells and human HeLa cells. Short term uptake was measured by a rapid sampling technique which allows sampling of cell suspensions in intervals as short as one and one-half seconds. The initial segments of the time courses were the same in untreated, wild-type cells in which uridine is rapidly phosphorylated and in cells in which uridine phosphorylation was prevented due to lack of ATP or uridine kinase. The initial rates of uptake, therefore, reflected the rate of uridine transport. Uridine uptake, however, was approximately linear for only five to ten seconds at uridine concentrations from 20–160 μM and somewhat longer at higher concentrations. In phosphorylating cells the rate of uridine uptake (at 80 μM) then decreased to about 20–30% of the initial rate and this rate was largely determined by the rate of phosphorylation rather than transport. At uridine concentrations below 1 μM, however, the rate of intracellular phosphorylation in Novikoff cells approached the transport rate. The apparent substrate saturation of phosphorylation suggests the presence of a low K_m uridine phosphorylation system in these cells. The “zero-trans” (zt) K_m for the facilitated transport of uridine as estimated from initial uptake rates fell between 50 and 240 μM for all cell lines examined. The zero-trans V_max values were also similar for all the lines (4–15 pmoles/μ1 cell H₂O.sec). The time courses of uridine uptake by CHO cells and the kinetic constants for transport were about the same whether the cells were propagated (and analyzed for uridine uptake) in suspension or monolayer culture. When Novikoff cells were preloaded with 10 μM uridine the apparent K_m and V_max values (infinite-trans) were two to three times higher than the corresponding zero-trans values. Uridine transport was inhibited in a simple competitive manner by several other ribo- and deoxyribonucleosides. All nucleosides seem to be transported by the same system, but with different efficiencies. Uridine transport was also inhibited by hypoxanthine, adenine, thymine, Persantin, papaverin, and o-nitrobenzylthioinosine, and by pretreatment of the cells with p-chloromercuri-benzoate, but not by high concentrations of cytosine, D-ribose or acronycin. The inhibition of uridine transport by Persantin involved changes in both V and K. Because of the rapidity of transport, some loss of intracellular uridine occurred when cells were rinsed in buffer solution to remove extracellular substrate, even at 0°. This loss was prevented by the presence of a transport inhibitor, Persantin, in the rinse fluid or by separating suspended cells from the medium by centrifugation through oil. Metabolic conversion of intracellular uridine were also found to continue during the rinse period. The extent of artifacts due to efflux and metabolism during rinsing increased with duration of the rinse.     

Growth rate of cultured Novikoff rat hepatoma cells as a function of the rate of thymidine and hypoxanthine transport.

Novikoff rat hepatoma cells were propagated in suspension culture in the presence of 1 micron methotrexate and various concentrations of hypoxanthine (or adenosine plus guanosine) and thymidine and with or without the inhibitor of nucleoside and purine transport, Persantin (dipyridamole). Methotrexate-treated cells failed to replicate and died even if the medium was supplemented with either thymidine or a purine source, but normal replication occurred when both were present. The additional presence of Persantin reduced the rate of transport of thymidine or hypoxanthine and thus their incorporation into the nucleotide pool and decreased the rate of cell replication. The growth rate of the cells was directly proportional to the rate of incorporation of thymidine (in the presence of excess hypoxanthine) or of hypoxanthine (in the presence of excess thymidine) until the normal maximum growth rate was obtained. Normal cell replication in the presence of methotrexate and Persantin occurred only when the medium was supplemented with 500 micron hypoxanthine and 30 micron thymidine. The results illustrate a dependence of the growth rate of mammalian cells on the rate of transport of essential nutrients into the cell.     

Facilitated transport of 6-mercaptopurine and 6-thioguanine and non-mediated permeation of 8-azaguanine in novikoff rat hepatoma cells and relationship to intracellular phosphoribosylation

6-Mercaptopurine and 6-thioguanine strongly inhibited the zero-trans entry of hypoxanthine into Novikoff rat hepatoma cells which lacked hypoxanthine/guanine phosphoribosyltransferase, whereas 8-azaguanine had no significant effect. 6-Mercaptopurine was transported by the hypoxanthine carrier with about the same efficiency as its natural substrates (Michaelis-Menten constant = 372 ± 23 μM; maximum velocity = 30 ± 0.7 pmol/μl cell H₂O per s). 8-Azaguanine entry into the cells, on the other hand, showed no sign of saturability and was not significantly affected by substrates of the hypoxanthine/guanine carrier. The rate of entry of 8-azaguanine at 10–100 μM amounted to only about 5% of that of hypoxanthine transport and was related to its lipid solubility in the same manner as observed for various substances whose permeation through the plasma membrane is believed to be non-mediated. Only the non-ionized form of 8-azaguanine (pK_a = 6.6) permeated the cell membrane.Studies with wild type Novikoff cells showed that permeation into the cell was the main rate-determining step in the conversion of extracellular 8-azaguanine to intracellular aza-GTP and its incorporation into nucleic acids. In contrast, 6-mercaptopurine was rapidly transported into cells and phosphoribosylated; the main rate-determining step in its incorporation into nucleic acids was the further conversion of 6-mercaptopurine riboside 5'-monophosphate.     

Activation of cultured bovine aortic endothelial cells by extracellular pyrimidine triphosphate.

1. The addition of ATP to cultured bovine aortic endothelial cells induced the increase in intracellular free calcium concentration ([Ca2+]i) and thereby activated the sodium/proton exchanger and the prostacyclin production in a similar dose-dependent manner, as observed by the addition of ATP. 2. Other nucleoside triphosphates also activated the cells and the potency orders of the nucleotides were ATP greater than UTP greater than ITP greater than CTP greater than GTP for all the responses. 3. Pretreatment of the cells with UTP desensitized the response to ATP and the pretreatment of ATP desensitized the response to UTP. 4. The responses to ATP and UTP were inhibited by neither pertussis nor cholera toxin. 5. The receptor for UTP, however, may be a distinct pyrimidinoceptor different from the purinoceptor of the cells for ATP, because the 50% effective concentration of UDP was much larger than that of UTP, while ATP and ADP were essentially equipotent ligands to the endothelial cells.     

Phenotypic conversion of cultured mouse embryo cells by aza pyrimidine nucleosides.

Cells of the $\text{C3H}\text{10}\text{T}\text{1}\text{2}\text{CL8}$ line, which are nonmyoblastic in nature, form functional myotubes when treated with low concentrations of 5-azacytidine. Further characterization of the myotubes revealed that they arise from the fusion of mononucleated precursors and not as a result of endoreplication. They accumulate histochemically detectable myosin ATPase activity as well as acetylcholine receptors capable of binding radioactively labeled α-bungarotoxin. The deoxy analog, 5-aza-2′-deoxycytidine, induced myogenic conversion at one-tenth of the maximally effective concentration of 5-azacytidine. The ability of both analogs to induce myotube formation and to cause cytotoxicity was strongly influenced by cotreatment with certain pyrimidine nucleosides. These effects were consistent with a requirement for metabolism of both aza compounds to phosphorylated derivatives and with a mechanism of action based on their incorporation into DNA. Concentrations of the analogs causing myogenic conversion did not substantially alter rates of DNA, RNA, or protein synthesis as measured by precursor incorporation into intact cells. The induction of myotubes by 5-azacytidine in cells synchronized by two different methods required that treatment with the analog was carried out at a critical phase early in S phase. Thus the mechanism of drug action appears to be linked to specific DNA synthesis.     

Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells.

Incubation of pig desoctapeptide-(B23-30)-insulin with trypsin in solvent systems consisting of dimethyl sulphoxide, butane-1,4-diol and Tris buffer resulted in the formation of an extra peptide bond between Arg-B22 and Gly-A1 in the DOPI molecule. This DOPI derivative can also be regarded as pig des-(23-63)-proinsulin. The structure of the new, previously unreported, proinsulin analogue was determined on the basis of amino acid analysis, dansylation and digestion with Staphylococcus aureus V8 proteinase. Receptor-binding ability of des-(23-63)-proinsulin was 20% of that of pig desoctapeptide-(B23-30)-insulin and 0.02% of that of pig insulin.     

Thymidine transport by Novikoff rat hepatoma cells synchronized by double hydroxyurea treatment

Suspension cultures of Novikoff rat hepatoma cells were synchronized by a double hydroxyurea block. About 80% of the cells of the population doubled 5 to 8 h after the reversal of the second hydroxyurea block. At all stages of the cell cycle, thymidine was rapidly incorporated into the acid-soluble pool of the cells (mainly dTTP) and the rate of incorporation was limited by the rate of thymidine transport. The rate of thymidine transport per cell roughly doubled during the S or late S phase and decreased again to the base level during cell division. This was reflected by corresponding changes in V_max for thymidine transport, whereas the apparent K_m remained constant throughout the cell cycle.     

Growth rate of cultured Novikoff rat hepatoma cells as a function of the rate of thymidine and hypoxanthine transport

Summary Novikoff rat hepatoma cells were propagated in suspension culture in the presence of 1m methotrexate and various concentrations of hypoxanthine (or adenosine plus guanosine) and thymidine and with or without the inhibitor of nucleoside and purine transport, Persantin (dipyridamole). Methotrexate-treated cells failed to replicate and died even if the medium was supplemented with either thymidine or a purine source, but normal replication occurred when both were present. The additional presence of Persantin reduced the rate of transport of thymidine or hypoxanthine and thus their incorporation into the nucleotide pool and decreased the rate of cell replication. The growth rate of the cells was directly proportional to the rate of incorporation of thymidine (in the presence of excess hypoxanthine) or of hypoxanthine (in the presence of excess thymidine) until the normal maximum growth rate was obtained. Normal cell replication in the presence of methotrexate and Persantin occurred only when the medium was supplemented with 500 m hypoxanthine and 30 m thymidine. The results illustrate a dependence of the growth rate of mammalian cells on the rate of transport of essential nutrients into the cell.