Clive Woffendin

Species differences in sensitivity of nucleoside transport in erythrocytes and cultured cells to inhibition by nitrobenzylthioinosine, dipyridamole, dilazep and lidoflazine

Differences in sensitivity of uridine transport in erythrocytes and cultured cells to inhibition by dipyridamole, dilazep and lidoflazine were largely species-specific; uridine transport in human cells, and probably in pig and rabbit cells, was 2-3- and 10-times more sensitive to inhibition by dipyridamole (IC50 approx. 50 nM) and about 10- and 20-times more sensitive to dilazep inhibition (IC50 approx. 5 nM) than transport in mouse and rat cells, respectively. Uridine transport in human erythrocytes and HeLa cells was strongly inhibited by lidoflazine (IC50 10-140 nM), whereas that in both mouse and rat cells was highly resistant (IC50 greater than 10 microM). Superimposed on species-specific differences were some cell type specific differences in sensitivity of nucleoside transport to these inhibitors. Uridine transport in Walker 256 rat carcinoma cells was more resistant to dipyridamole and dilazep than that of other rat cells. Transport in human Hep-2 cells was more resistant to lidoflazine (IC50 2000 nM) than that of human erythrocytes and HeLa cells, whereas it showed similar sensitivity to dilazep and dipyridamole. Uridine transport in Chinese hamster cells was also more resistant to dilazep than that of baby hamster kidney cells. In addition HeLa cells and clones thereof expressed uridine transporters (about 50% each) with difference of about 1000-fold in sensitivity to inhibition by dilazep (IC50 approx. 5 nM and 5 microM, respectively).

Na+-dependent and -independent transport of uridine and its phosphorylation in mouse spleen cells

Rapid kinetic techniques were used to study the transport and salvage of uridine and other nucleosides in mouse spleen cells. Spleen cells express two nucleoside transport systems: (1) the non-concentrative, symmetrical, Na+-independent transporter with broad substrate specificity, which has been found in all mammalian cells and is sensitive to inhibition by dipyridamole and nitrobenzylthioinosine; and (2) a Na+-dependent nucleoside transport, which is specific for uridine and purine nucleosides and resistant to inhibition by dipyridamole and nitrobenzylthioinosine. The kinetic properties of the two transporters were determined by measuring uridine influx in ATP-depleted cells and dipyridamole-treated cells, respectively. The Michaelis-Menten constants for Na+-independent and -dependent transport were about 40 and 200 microM, respectively, but the first-order rate constants were about the same for both transport systems. Nitrobenzylthioinosine-sensitivity of the facilitated nucleoside transporter correlated with the presence of about 10,000 high-affinity (Kd = 0.6 nM) nitrobenzylthioinosine-binding sites per cell. The turnover number of the nitrobenzylthioinosine-sensitive nucleoside transporter was comparable to that of mouse P388 leukemia cells. The activation energy of this transporter was 20 kcal/mol. Entry of uridine via either of the transport routes was rapidly followed by its phosphorylation and conversion to UTP. The Michaelis-Menten constant for the in situ phosphorylation of uridine was about 50 microM and the first-order rate constants for phosphorylation and transport were about the same. The spleen cells also efficiently salvaged adenosine, adenine, and hypoxanthine, but not thymidine.

Use of formycin B as a general substrate for measuring facilitated nucleoside transport in mammalian cells

Formycin B, a C-nucleoside analog of inosine, is not catabolized by human erythrocytes and mouse P388 leukemia cells and is only very inefficiently phosphorylated in these cells. This relative inertness allows the measurement of its transport into and out of the cells uncomplicated by metabolic conversions. We have measured the zero-trans and equilibrium exchange flux of formycin B in these cells by rapid kinetic techniques. The Michaelis-Menten constants and maximum velocities for formycin B transport in both types of cell were similar to those previously reported for uridine and thymidine. Nevertheless, the differential mobility of the substrate-loaded and empty carrier of human erythrocytes was less for formycin B than uridine as substrate. Formycin B influx was inhibited by other nucleosides in accordance with their affinities for the carrier, but unaffected by purines. The inhibition of formycin B influx by nitrobenzylthioinosine and dipyridamole was also identical to that observed with uridine as substrate (IC50 = 10 and 30 nM, respectively). Formycin B accumulated in both types of cell to 30-40% higher concentrations than were present in the medium. This concentrative accumulation was not due to active transport, metabolism or partitioning into membrane lipids. It seems to reflect binding of formycin B to intracellular components, but does not interfere significantly with measurements of its transport.

Na+-dependent, active and Na+-independent, facilitated transport of formycin B in mouse spleen lymphocytes

Na(+)-dependent, active and Na(+)-independent facilitated nucleoside transport were characterized in mouse spleen cells using rapid kinetic techniques and formycin B, a metabolically inert analog of inosine, as substrate. The Michaelis-Menten constants for formycin B transport by the two transporters were about 30 and 400 microM, respectively. The first-order rate constant for Na(+)-dependent transport was about 4-times higher than that for facilitated formycin B transport. The Na(+)-dependent carrier is specific for uridine and purine nucleosides and accumulates formycin B concentratively in an unmodified form. Concentrative accumulation was inhibited by ATP depletion and gramicidin and ouabain treatment of the cells. Our data indicate a single Na(+)-binding site on the Na(+)-dependent nucleoside carrier and a Michaelis-Menten constant for Na+ of about 10 mM. This transporter was not significantly inhibited by dipyridamole and nitrobenzylthioinosine, inhibitors of the facilitated transporter. The Na(+)-independent, facilitated nucleoside transporter of spleen cells exhibits properties comparable to those of the carriers present in mammalian cells in general. The B lymphocytes remaining after depletion of spleen cell populations of T lymphocytes by incubation with a combination of T-cell specific monoclonal antibodies plus complement exhibited about the same activities of active and facilitated nucleoside transport as the original suspension.

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.

Nucleoside transporter of pig erythrocytes. Kinetic properties, isolation and reaction with nitrobenzylthioinosine and dipyridamole

Rapid kinetic techniques were used to measure the transport of uridine in pig erythrocytes in zero-trans entry and exit and equilibrium exchange protocols. The kinetic parameters were computed by fitting appropriate integrated rate equations to the time-courses of transmembrane equilibration of radiolabeled uridine. Transport of uridine conformed to the simple carrier model with directional symmetry, but differential mobility of substrate-loaded and empty carrier. At 5 degrees C, the carrier moved about 30-times faster when loaded than when empty. Uridine transport was inhibited in a concentration-dependent manner by nitrobenzylthioinosine and dipyridamole and the inhibition correlated with the binding of the inhibitors to high-affinity binding sites on the cells (Kd about 1 and 10 nM, respectively). Thus, in its kinetic properties, differential mobility when empty and loaded, and sensitivity to inhibition by nitrobenzylthioinosine and dipyridamole, the transporter of pig erythrocytes is very similar to that of human erythrocytes. Also, the total number of high-affinity binding sites for nitrobenzylthioinosine and dipyridamole/cell were similar for the two cell types and the [3H]nitrobenzylthioinosine-labeled carrier of pig erythrocytes, just as that of human red cells, was mainly recovered in the band 4.5 protein fraction of Triton X-100-solubilized membranes. However, sodium dodecylsulfate-polyacrylamide gel electrophoresis of photoaffinity-labeled band 4.5 membrane proteins indicated a slightly higher molecular weight for the transporter from pig than human erythrocytes. We have also confirmed the lack of functional sugar transport in erythrocytes from adult pigs by measuring the uptake of various radiolabeled sugars. But in spite of the lack of functional sugar transport we recovered as much band 4.5 protein from pig as from human erythrocyte membranes.

Comparison of the equilibrium exchange of nucleosides and 3-O-methylglucose in human erythrocytes and of the effects of cytochalasin B, phloretin and dipyridamole on their transport

Because of similarities in the physical and molecular properties of the nucleoside and sugar transporters of human erythrocytes and the photoaffinity labeling of the sugar transporter by 8-azidoadenosine (Jarvis et al. (1986) J. Biol. Chem. 261, 11077-11085), we have directly compared the equilibrium exchange of uridine and 3-O-methylglucose in these cells as measured by rapid kinetic techniques under identical experimental conditions. Both the Michaelis-Menten constant and maximum velocity were about 100-fold higher for 3-O-methylglucose exchange than for uridine exchange so that the first order rate constants for both transporters were about the same. When calculated on the basis of the number of nucleoside and sugar carriers per red cell estimated by equilibrium binding of nitrobenzylthioinosine and cytochalasin B, respectively, the turnover numbers for the sugar and nucleoside carriers with 3-O-methylglucose and uridine, respectively, as substrates were quite similar. Various sugars up to concentrations of 108 mM had no effect on the exchange of 500 microM uridine or adenosine, and uridine up to a concentration of 50 mM had no effect on the exchange of 10 mM 3-O-methylglucose. Adenosine, on the other hand, inhibited 3-O-methylglucose exchange in a concentration dependent manner, though not very effectively (IC50 approximately equal to 3 mM). Both uridine and 3-O-methylglucose exchange were inhibited in a concentration dependent manner by cytochalasin B, phloretin and dipyridamole, but cytochalasin B and phloretin were 100-times more effective in inhibiting 3-O-methylglucose than uridine exchange, whereas the opposite was the case for the inhibition by dipyridamole.

Interaction of [3H]dipyridamole with the nucleoside transporters of human erythrocytes and cultured animal cells.

SummaryEquilibrium binding of [3H]dipyridamole identified high-affinity (Ki∼10nm) binding sites on human erythrocytes (∼5×105 sites/cell) and on HeLa cells (∼5×106 sites/cell). The equilibration of dipyridamole with these sites on human erythrocytes was compatible with a second-order process which proceeded at 22°C with a rate constant of about 6×106m−1 sec−1. Binding of dipyridamole to these sites correlated kinetically with the inhibition of the equilibrium exchange of 500 μm uridine in these cells and was inhibited in a concentration-dependent manner by nucleosides and other inhibitors of nucleoside transport, such as nitrobenzylthioinosine, dilazep and lidoflazine, but not by hypoxanthine, which is not a substrate for the nucleoside transporter of human erythrocytes. The results indicate that the substrate binding site of the transporter is part of the high-affinity dipyridamole binding site. Bound [3H]dipyridamole became displaced from these sites on human erythrocytes by incubation with an excess of unlabeled dipyridamole or high concentrations of nucleosides and inhibitors of nucleoside transport, but neither by hypoxanthine nor sugars. Dissociation of [3H]dipyridamole behaved as a simple first-order process, but the rate constant was about one order of magnitude lower (about 3×10−3 sec−1) than anticipated for typical ligand-protein binding on the basis of the measured association rate and equilibrium constants. The reason for this discrepancy has not been resolved. No high-affinity dipyridamole binding sites were detected on Novikoff rat hepatoma cells, P388, L1210 and S49 mouse leukemia cells or Chinese hamster ovary cells, and their absence correlated with a greater resistance of nucleoside transport in these cells to inhibition by dipyridamole. All cells expressed considerable low affinity (Kd>0.5 μm) and nonspecific binding of dipyridamole.

Effects of Ca2+-channel antagonists on nucleoside and nucleobase transport in human erythrocytes and cultured mammalian cells

Lidoflazine strongly inhibited the equilibrium exchange of uridine in human erythrocytes (Ki approximately 16 nM). Uridine zero-trans influx was similarly inhibited by lidoflazine in cultured HeLa cells (IC50 approximately to 80 nM), whereas P388 mouse leukemia and Novikoff rat hepatoma cells were three orders of magnitude more resistant (IC50 greater than 50 microM). Uridine transport was also inhibited by nifedipine, verapamil, diltiazem, prenylamine and trifluoperazine, but only at similarly high concentrations in both human erythrocytes and the cell lines. IC50 values ranged from about 10 microM for nifedipine and about 20 microM for verapamil to more than 100 microM for diltiazem, prenylamine and trifluoperazine. The concentrations required for inhibition of nucleoside transport are several orders higher than those blocking Ca2+ channels. Lidoflazine competitively inhibited the binding of nitrobenzylthioinosine to high-affinity sites in human erythrocytes, but did not inhibit the dissociation of nitrobenzylthioinosine from these sites on the transporter as is observed with dipyridamole and dilazep.

Nucleoside and nucleobase transport and metabolism in wild type and nucleoside transport-deficient Aedes albopictus cells.

Nucleoside and nucleobase transport and metabolism were measured in ATP-depleted and normal Aedes albopictus mosquito cells (line C-7-10) by rapid kinetic techniques. The cells possess a facilitated diffusion system for nucleosides, which in its broad substrate specificity and kinetic properties resembles that present in many types of mammalian cells. The Michaelis-Menten constant for uridine transport at 28 degrees C is about 180 microM. However, the nucleoside transporter of the mosquito cells is resistant to inhibition by nmolar concentrations of nitrobenzylthioinosine and the cells lack high affinity nitrobenzylthioinosine binding sites. The cells also possess an adenine transporter, which is distinct from the nucleoside transporter. They lack, however, a hypoxanthine transport system and are deficient in hypoxanthine phosphoribosyltransferase activity, which explains their failure to efficiently salvage hypoxanthine from the medium. The cells possess uridine and thymidine phosphorylase activities and, in contrast to cultured mammalian cells, efficiently convert uracil to nucleotides. An adenosine-resistant variant (CAE-3-6) of the C-7-10 cell line is devoid of significant nucleoside transport activity but transports adenine normally. Residual entry of various nucleosides into these cells and of hypoxanthine and cytosine into wild type and mutant cells is strictly non-mediated. The rate of permeation of various nucleosides and of hypoxanthine into the CAE-3-6 cells is related to their hydrophobicity. Uridine permeation into CAE-3-6 cells exhibits an activation energy of about 20 kcal/mol. At high uridine concentrations permeation is sufficiently rapid to partly overcome the limitation in nucleoside salvage imposed by the nucleoside transport defect in these cells.