John Erbe

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

Abstract 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.

Membrane effects of cytochalasin B. Competitive inhibition of facilitated diffusion processes in rat hepatoma cells and other cell lines and effect on formation of functional transport sites.

SummaryCytochalasin B competitively inhibits the transport of 2-deoxy-d-glucose and thymidine in a number of different cell lines (Novikoff rat hepatoma cells, mouse L, S180 and Ki-MSV-transformed BALB/3T3 cells, and human HeLa cells). The apparentKm values for the transport of these substrates as well as the apparentKi values for the inhibition by cytochalasin B are very similar for the various cell lines, and the effect is readily and completely reversed by removal of the chemical. Thymidine transport by Chinese hamster ovary cells however, is little affected by cytochalasin B, whereas the transport of 2-deoxy-d-glucose, uridine and guanine by these cells is competitively inhibited to about the same extent as in other cell lines. In addition and concomitant with the inhibition of cytokinesis and an alteration in cell shape, cytochalasin B also impairs and delays the formation of functional transport sites for thymidine, guanine and choline in synchronized populations of Novikoff cells without affecting the apparent affinities of the transport systems for their substrates. This effect is unrelated to the direct inhibition of the transport processes, since the drug does not directly inhibit choline transport and has no effect on the formation of 2-deoxy-d-glucose transport sites in spite of the fact that it strongly inhibits the transport of this substrate. The inhibition of functional transport sites may be due to the induction of a structural alteration in the membrane by cytochalasin B which impairs the insertion of new proteins of certain but not all transport systems into the membrane.

Glucocorticoids--uptake by simple diffusion by cultured Reuber and Novikoff rat hepatoma cells.

Abstract The initial rate of uptake of [ 3 H] prednisolonc by cultured NovikolT and Reuber rat hepatoma cells at 18 was directly proportional to the hormone concentration in the medium between 0.01 μm and 5 mM. Uptake of 0.05 μM prednisolone was not affected by 100 μM dexamethasone or deoxycorticosterone or by depletion of the cells of ATP by proincubation in glucose-free medium containing KCN and iodoacetate. Uptake was also not affected by treatment of the cells with neuraminidase or phospholipase C and no countertransport of prednisolone could be demonstrated. Uptake was also unaffected by a 5000-fold excess of d -glucose in the medium, in spite of the fact that prednisolone acts as a simple competitive inhibitor of d -glucosc transport. The results indicate that prednisolone is not taken up by the d -glucosc or some glucocorticoid-specific transport system, but rather enters both types of cells by simple diffusion. Net uptake of prednisolone by the glucocorticoid-unresponsive Novikoff cells ceased when the intracellular concentration was 20–100% higher than that in the medium regardless of the medium concentration, indicating some nonspecific binding of the hormone to cellular components. At a concentration of 0.01 μM in the medium the glucocorticoid-responsive Reuber hcpatoma cells accumulated two to three times more prednisolone/cell than Novikoff cells. This concentrative effect diminished at higher concentrations, indicating saturation of binding sites.

Facilitated transport of inosine and uridine in cultured mammalian cells is independent of nucleoside phosphorylases

The zero-trans uptake of uniformly and base-labeled inosine and uridine was measured a 25 degrees C in suspensions of Novikoff rat hepatoma cells, Chinese hamster ovary cells, mouse L cells, mouse S49 lymphoma cells and a purine-nucleoside phosphorylase-deficient subline thereof (NSU-1), and in monolayer culture of mouse 3T3 and L cells. The initial velocities of uptake of both nucleosides were about the same in all cell lines investigated, regardless of the position of the label or of the substrate concentration between 3 and 300 microM or whether or not the cells possessed uridine or purine-nucleoside phosphorylase activity. The kinetic parameters for the facilitated transport of uridine and inosine were also similar in phosphorylase positive and negative cell lines (K = 120--260 microM and V = 6--40 pmol/microliters cell water per s) and the transport activities of the cells exceeded their total phosphorylase activities by at least 10-fold for uridine and 1--2-fold for inosine. Chromatographic fractionation of the intracellular contents and of the culture fluid showed that the free nucleosides appeared intracellularly prior to and more rapidly than their phosphorolysis products. During the initial 20--60 s of uptake of U-14C-labeled nucleosides the rates of intracellular appearance of ribose-1-P and base were about the same. After several minutes of incubation, on the other hand, the main intracellular component was ribose-1-P whereas the base attained a low intracellular steady-state concentration and accumulated in the medium due to exit transport. Other nucleosides, dipyridamole and nitrobenzylthioinosine, specifically inhibited the transport of uridine and inosine, and depressed the intracellular accumulation of ribose-1-P and the formation of base commensurate with that inhibition. The data indicate that the metabolism of inosine and uridine by the various cell lines can be entirely accounted for by the facilitated transport of unmodified nucleoside into the cell followed by intracellular phosphorolysis.

Temperature-dependent changes in activation energies of the transport systems for nucleosides, choline and deoxyglucose of cultured Novikoff rat hepatoma cells and effects of cytochalasin B and lipid solvents

SummaryThe initial rates of transport of uridine, thymidine, purines, choline and 2-deoxy-d-glucose by cultured Novikoff rat hepatoma cells were determined as a function of temperature between 5 and 41°C. Arrhenius plots of all transport systems exhibited sharp breaks in slope; between 17 and 23° for uridine, thymidine and hypoxanthine-guanine transport and between 29 and 32° for choline and 2-deoxy-d-glucose transport. The activation energies for the transport systems changed from 15–26 kcal/mole below the transition temperatures to 4–9 kcal/mole above the transition temperatures. Propagation of the cells in the presence ofcis-6-octadecenoic acid which results in marked changes in the lipid composition of cell membrane, had little effect on the temperature characteristics of the various transport systems. Similarly, propagation of the cells for 24 hr in media containing Tween 40 or nystatin had no effect on the capacity of the cells to transport the various substrates or on the temperature dependence of the transport systems. The presence of ethanol, phenethyl alcohol or Persantin at concentrations that inhibited thymidine and 2-deoxy-d-glucose transport between 40 and 70% also did not alter the transition temperatures or activation energies for the transport of these substrates. Cytochalasin B, on the other hand, shifted the transition temperature for 2-deoxy-d-glucose transport to higher temperatures in a concentration-dependent manner, whereas it had no effect on the temperature dependence of thymidine transport.

Inhibition of transport systems in cultured rat hepatoma cells by colcemid and ethanol.

Abstract The transport of uridine, hypoxanthine, and choline in cultures of Novikoff rat hepatoma cells is competitively inhibited by colcemid with apparent K i values of 135, 60, and 250 μM respectively, whereas the transport of 2-deoxy-D-glucose is not affected. Ethanol at high concentrations inhibits the transport of all four substrates in an apparent competitive manner.