Peter G.W. Plagemann

Permeation of Nucleosides, Nucleic Acid Bases, and Nucleotides in Animal Cells

Publisher Summary This chapter discusses the importance of the mechanism of permeation of nucleosides, nucleobases, and nucleotides through the cell membranes of eukaryotes. Some of the reasons of its importance include (1) many anticancer and immunosuppressive agents presently in use or under development are nucleoside, nucleotide, or nucleobase analogs and a clear understanding of their mode of entry into cells and metabolism is important in the assessment of their mode of action, efficacy, and optimal administration, and (2) radioactively labeled nucleosides and nucleic acid bases are widely used as precursors to label specifically the nucleic acids of various types of organisms or of the viruses or plasmids replicating therein as well as to assess the rates of nucleic acid synthesis. An interpretation of the rates of nucleoside and base incorporation into nucleic acids, be it RNA or DNA, depends on a clear understanding of the extent to which these rates may reflect the rates of the conversion of the extracellular substrate to intracellular nucleotides, which are the direct precursors in nucleic acid synthesis. Elimination of the ambiguities inherent in metabolizing cells is of clear advantage to transport studies. Nucleoside and purine transport have been studied successfully in the absence of intracellular metabolism by the use of erythrocytes or of mutant clones of cultured animal cells that are deficient in specific metabolic enzymes and by the use of cell/substrate systems in which substrate metabolism is blocked in some other manner. The chapter also discusses the carrier model for facilitated diffusion and tests for its applicability to nucleoside and base transport.

The roles of transport and phosphorylation in nutrient uptake in cultured animal cells.

Publisher Summary Three main classes of nutrients—nucleosides, nucleobases, and hexoses (and possibly a fourth, the water-soluble vitamins)—are taken up into cultured cells as a result of the tandem action of a nonconcentrative transport system and an intracellular enzyme that introduces into the transported substrate an anionic group thus confering impermeability. For several substrates representing the first three of these nutrient classes, both members of the uptake pathway are characterized kinetically: (1) the transport systems with cells and substrates that, by virtue of enzyme deficiency, adenosine triphosphate (ATP) depletion, or chemical design, are metabolically inert and (2) the enzymes, as purified proteins, in idealized milieu. The consequences of changes in substrate concentration, in temperature, and in the effective kinetic parameters of transport and phosphorylation on rates of uptake demonstrate the complexity of this interdependence and the errors of interpretation that can accrue if the complexity is overlooked. Flux in a tandem pathway follows a biphasic time course. The first phase corresponds to the attainment of steady-state levels of free intracellular substrate. The second phase represents steady-state flux through the pathway—that is, the rate of accumulation of impermeable product.

Chapter 16 A Rapid-Mixing Technique to Measure Transport in Suspended Animal Cells: Applications to Nucleoside Transport in Novikpff Rat Hepatoma Cells

Publisher Summary The chapter discusses techniques, which permit an operational separation of transport and metabolism. This separation can be achieved by genetic, chemical, or kinetic manipulation, or a combination thereof. The transport of various compounds across mammalian cell membranes is frequently found to occur with a rapidity which necessitates collecting data at intervals of a few seconds. By means of a dual-syringe device, suspended cells can be mixed nearly instantaneously with radioactively labeled substrate and separated from the substrate again within seconds by centrifugation into silicone oil. Depending on the cell-substrate system under investigation, initial transport velocities may be either measured directly or calculated from the time course with which equilibrium across the membrane is attained. With nonmetabolizing systems, the dual-syringe apparatus is adaptable to a variety of experimental protocols-zero-trans, equilibrium exchange, and infinite-cis—which in combination make possible a thorough kinetic characterization of a transport system.

Replication of lactate dehydrogenase-elevating virus in macrophages. 1. Evidence for cytocidal replication.

Cultures of starch-elicited peritoneal mouse macrophages in medium containing macrophage growth factor (MGF) were infected with lactate dehydrogenase-elevating virus (LDV) and, after various times in culture, LDV production was monitored as a function of time by infectivity titrations in mice, by measuring [3H]uridine incorporation into LDV RNA and extracellular LDV, by autoradiographic analysis of the proportion of productively infected cells and by electron microscopy. Regardless of the age of the cultures when infected with LDV, only a small proportion of the macrophages (generally between 3 and 20% of the total) became productively infected after a primary infection; maximum virus RNA synthesis and virus production occurred during the first 24 h after infection and then decreased precipitously. Productively infected macrophages could be readily recognized in electron micrographs of 24-h infected macrophage cultures and in sections of spleens from 24-h infected mice by characteristic morphological alterations. These consisted of formation of clusters of double-membrane vesicles with a diameter of 100 to 300 mumol, budding of nucleocapsids into vesicles with single membranes and accumulation of mature virions in these vesicles. One to 4 days later, however, such cells were no longer found in infected cultures or spleens of infected mice and superinfection did not restimulate LDV replication. Cultures established with macrophages from 1-day LDV-infected mice also did not support LDV replication. We conclude that LDV replication in cultures or mice is limited to a single cycle in a subpopulation of macrophages and that infection leads to cell death and rapid phagocytosis of the dead cells by the resistant, uninfected macrophages.

Nucleoside transport in cultured mammalian cells multiple forms with different sensitivity to inhibition by nitrobenzylthioinosine or hypoxanthine

The zero-trans influx of 500 microM uridine by CHO, P388, L1210 and L929 cells was inhibited by nitrobenzylthioinosine ( NBTI ) in a biphasic manner; 60-70% of total uridine influx by CHO cells and about 90% of that in P388, L1210 and L929 cells was inhibited by nmolar concentrations of NBTI (ID50 = 3-10 nM) and is designated NBTI -sensitive transport. The residual transport activity, designated NBTI -resistant transport, was inhibited by NBTI only at concentrations above 1 microM (ID50 = 10-50 microM). S49 cells exhibited only NBTI -sensitive uridine transport, whereas Novikoff cells exhibited only NBTI -resistant uridine transport. In all instances NBTI -sensitive transport correlated with the presence of between 7 7 X 10(4) and 7 X 10(5) high-affinity NBTI binding sites/cell (Kd = 0.3-1 nM). Novikoff cells lacked such sites. The two types of nucleoside transport, NBTI -resistant and NBTI -sensitive, were indistinguishable in substrate affinity, temperature dependence, substrate specificity, inhibition by structurally unrelated substances, such as dipyridamole or papaverine, and inhibition by sulfhydryl reagents or hypoxanthine. We suggest, therefore, that a single nucleoside transporter can exist in an NBTI -sensitive and an NBTI -resistant form depending on its disposition in the plasma membrane. The sensitive form expresses a high-affinity NBTI binding site(s) which is probably made up of the substrate binding site plus a hydrophobic region which interacts with the lipophilic nitrobenzyl group of NBTI . The latter site seems to be unavailable in NBTI -resistant transporters. The proportion of NBTI -resistant and sensitive uridine transport was constant during proportion of NBTI -resistant and sensitive uridine transport was constant during progression of P388 cells through the cell cycle and independent of the growth stage of the cells in culture. There were additional differences in uridine transport between cell lines which, however, did not correlate with NBTI sensitivity and might be related to the species origin of the cells. Uridine transport in Novikoff cells was more sensitive to inhibition by dipyridamole and papaverine than that in all other cell lines tested, whereas uridine transport in CHO cells was the most sensitive to inactivation by sulfhydryl reagents.

Nucleoside transport by Novikoff rat hepatoma cells growing in suspension culture: Specificity and mechanism of transport reactions and relationship to nucleoside incorporation into nucleic acids

The transport of adenosine, guanosine, inosine, uridine and cytidine by Novikoff rat hepatoma cells (subline N1S1-67) is competitively inhibited by each of the other nucleosides, thymidine, persantin and phenethyl alcohol. Comparisons of the transport kinetics of the various nucleosides (Km and vmax), of the Km/Ki ratios for the inhibitions and of the effect of heat shock (47.5°, 5 min) on nucleoside transport suggest that guanosine and inosine are transported by a single system, whereas different specific systems transport adenosine and the pyrimidine nucleosides. Uridine and cytidine also seem to be transported by a single system. All systems are inactivated to about the same extent by treatment of the cells with p-chloromercuribenzoate. Incubation of the cells in media containing 2% trypsin or chymotrypsin, 1% neuraminidase or 0.025% phospholipase C for 15 min has no significant effect on uridine transport, and energy poisons have an effect only at relatively high concentrations. The incorporation of each of the nucleosides into nucleic acids by the cells also follows simple Michaelis-Menten kinetics and the apparent Km for each nucleoside is similar to that of its transport into the cell. Competitive inhibition of nucleoside transport by heterologous nucleosides, persantin or phenethyl alcohol results in an apparent competitive inhibition of nucleoside incorporation into nucleic acids and the apparent Ki values for the inhibitions are similar for both processes. The results suggest that the rate-limiting step in the incorporation of each of the nucleosides into nucleic acids is its transport into the cell.

Antibody response of mice to lactate dehydrogenase-elevating virus during infection and immunization with inactivated virus

BALB/c and Swiss mice were infected with lactate dehydrogenase-elevating virus (LDV) or immunized with glutaraldehyde-inactivated or ether-extracted virus and their plasma was monitored for anti-LDV IgG and IgM levels by ELISA and indirect fluorescent antibody staining, for neutralizing antibodies, for sensitized antibody-virus complexes, for immune complexes, and for total plasma IgG and IgM. In infected mice, anti-LDV IgM was transiently formed during the first 2 weeks post infection (p.i.) but only at a low level. Anti-LDV IgG was produced in a biphasic manner with an initial peak at about 10 days p.i. and a secondary rise reaching a maximum level 30-80 days p.i. which was retained throughout the persistent phase of infection. The concomitant appearance of comparable levels of low molecular weight immune complexes suggests that most anti-LDV IgG was complexed with LDV proteins. Also, as early as 10 days p.i., infectious antibody-LDV complexes developed, which were neutralizable by rabbit anti-mouse IgG, whereas antibodies that neutralize the infectivity of exogenously added LDV appeared only 1-2 months p.i. Throughout infection, most of the anti-LDV IgG was directed to VP-3, the envelope glycoprotein of LDV, which was found to exist in at least 10 distinct forms ranging in molecular weight from 24 to 42 kDa. Anti-LDV IgG levels as high as those observed in infected mice developed in mice immunized with inactivated LDV. Antibodies to glutaraldehyde-inactivated LDV were also mainly directed to VP-3, but exhibited no neutralizing activity. The polyclonal B cell activation associated with a persistent LDV infection and the formation of immune complexes were not observed in mice immunized with inactivated virus.

Replication of lactate dehydrogenase-elevating virus in macrophages. 2. Mechanism of persistent infection in mice and cell culture.

SUMMARY A primary infection of peritoneal macrophage cultures with the lactate dehydrogenase-elevating virus (LDV) results in productive infection of 3 to 20% of the cells. When cultures were incubated in the absence of macrophage growth factor (MGF), LDV production ceased after a single cycle, but in cultures in which macrophage replication was stimulated by the presence of MGF LDV production continued for several weeks at a low level, representing not more than 1% of that observed during the acute phase. Significant amounts of interferon were not present in either acutely or persistently infected cultures, and treatment of persistently infected cultures with anti-interferon globulin or superinfection with LDV did not significantly stimulate LDV replication. Macrophage cultures established with peritoneal macrophages from LDV-infected mice also showed only a low level of LDV replication and were resistant to superinfection by LDV. Mouse hepatitis virus, Semliki Forest virus and vesicular stomatitis virus, on the other hand, replicated normally in LDV-persistently infected macrophage cultures. LDV replication was relatively resistant to interferon whether added to the cultures or generated endogenously by infection with Newcastle disease virus or defective-interfering (DI) particles of vesicular stomatitis virus. Temperature-sensitive mutants or DI particles of LDV were not detected in LDV-persistently infected cultures or chronically infected mice. The results support our hypothesis that the decrease in LDV production in mice or macrophage cultures at the end of the acute phase results from the destruction of the subpopulation of macrophages that is permissive for LDV, and that the low level persistent infection involves the passage of the virus to new permissive cells that are generated continuously, although at a low rate, from non-permissive precursor cells.

Choline metabolism and membrane formation in rat hepatoma cells grown in suspension culture: I. Incorporation of choline into phosphatidylcholine of mitochondria and other membranous structures and effect of metabolic inhibitors☆

Abstract The synthesis of phosphatidylcholine and its corporation into membranes of Novikoff rat hepatoma cells (strain N1S1–67) cultivated in suspension culture was investigated using 3H-methyl-choline as specific precursor. When cells were suspended in basal medium containing 0.01 m m choline, the latter was taken up by the cells very rapidly and accumulated intracellularly bound in phosphorylcholine. Results from pulse-chase experiments have shown that phosphorylcholine is a precursor in the synthesis of phosphatidylcholine, probably via the Kennedy pathway, but cytidine diphosphate choline did not accumulate in detectable amounts. The synthesis of phosphorylcholine proceeded 4–5 times more rapidly than its subsequent incorporation into phosphatidylcholine and the cells (1.8 × 106 cells per ml) removed over 90% of the choline from the medium within 5–6 hr of incubation at 37 °. The rate of synthesis of phosphatidylcholine by cells was influenced to some extent by the stage of growth of the culture from which the cells were obtained, but was independent of the concentration of choline in the medium between 0.01 and 0.06 m m or the pH between 6.8 and 8.0. Newly synthesized phosphatidylcholine was detectable in cells only as an integral part of cellular membranes. Results from experiments involving differential centrifugation of cell homogenates and isopycnic sucrose density gradient centrifugation of cytoplasmic extracts indicate that about 15% of the total phosphatidylcholine synthesized was associated with the nuclei, 40–50% with the mitochondria, and the remainder with fragments of the plasma membrane and other membranous components with densities (1.13 – 1.15 g/cm3) lower than that of mitochondria (1.18 g/cm3). The incorporation of choline into the various membranous structures of the cell followed a similar time course. The density of the mitochondria and other membranous structures was approximately the same whether the cells were incubated in basal medium containing 0.01 or 0.10 m m choline. Inhibition of protein synthesis by treatment of cells with actidione or chloramphenicol did not affect the rates of incorporation of choline into phosphorylcholine or phosphatidylcholine for at least 3 hr. Both processes were slightly inhibited by treating cells with puromycin or actinomycin D.

Properties of the thymidine transport system of chinese hamster ovary cells as probed by nitrobenzylthioinosine

SummaryThe transport of thymidine into Chinese hamster ovary cells grown in suspension culture was measured under conditions in which thymidine was not metabolized, namely, when cells had been depleted of ATP. The system transporting thymidine was saturable (Kmzt=70μM), rapid (50% of transmembrane equilibrium level attained within 8 sec), and was apparently shared by other nucleosides, but not thymine or hypoxanthine. 6([4-nitrobenzyl]thio)-9-β-d-ribofuranosylpurine, “nitrobenzylthioinosine”, inhibited thymidine transport in a simple, noncompetitive fashion with an apparentKi=1.0 nM (based on total concentration of inhibitor, which significantly overestimates that of free inhibitor). The rate of expression of inhibition was slow (t1/2=17 sec) relative to the rate of association of thymidine with its transporter, and thymidine partially protected the transport system against inhibition by nitrobenzylthioinosine. The dissociation constant for the inhibitortransporter complex was estimated at about 0.1 nM, and the number of binding sites per cell at about 6×104. HeLa, P388 murine leukemia, and mouse L cells were as sensitive to nitrobenzylthioinosine inhibition of thymidine transport as Chinese hamster ovary cells; Novikoff rat hepatoma cells were much less sensitive.