Nucleoside transport in cultured LLC-PK1 epithelia.

The transport of nucleosides by LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, was characterised. Uridine influx was saturable (apparent Km approximately 34 microM at 22 degrees C) and inhibited by greater than 95% by nitrobenzylthioinosine (NBMPR), dilazep and a variety of purine and pyrimidine nucleosides. In contrast to other cultured animal cells, the NBMPR-sensitive nucleoside transporter in LLC-PK1 cells exhibited both a high affinity for cytidine (apparent Ki approximately 65 microM for influx) and differential 'mobility' of the carrier (the kinetic parameters of equilibrium exchange of formycin B are greater than those for formycin B influx). An additional minor component of sodium-dependent uridine influx in LLC-PK1 cells became detectable when the NBMPR-sensitive nucleoside transporter was blocked by the presence of 10 microM NBMPR. This active transport system was inhibited by adenosine, inosine and guanosine but thymidine and cytidine were without effect, inhibition properties identical to the N1 sodium-dependent nucleoside carrier in bovine renal outer cortical brush-border membrane vesicles (Williams and Jarvis (1991) Biochem. J. 274, 27-33). Late proximal tubule brush-border membrane vesicles of porcine kidney were shown to have a much reduced Na(+)-dependent uridine uptake activity compared to early proximal tubule porcine brush-border membrane vesicles. These results, together with the recent suggestion of the late proximal tubular origin of LLC-PK1 cells, suggest that in vivo nucleoside transport across the late proximal tubule cell may proceed mainly via a facilitated-diffusion process.     

Transport mechanisms in isolated plasma membranes. Nucleoside processing by membrane vesicles from mouse fibroblast cells grown in defined medium.

Plasma membrane vesicles were isolated from a subline of L929 mouse fibroblasts grown on defined medium in the absence of serum. These vesicles were not significantly contaminated by mitochondria or endoplasmic reticulum. The isolation procedure, a modification of that originally developed by McKeel and Jarett (McKeel, D.W., and Jarett, L. (1970) J. Cell Biol. 44, 417-432) employs mechanical homogenization in isotonic medium followed by differential centrifugation. The resultant plasma membrane vesicles take up radioactivity when exposed to uniformly labeled nucleosides. Two subfractions of the plasma membrane were isolated, distinguished by their differing activity of 5'-nucleotidase and (Na+,K+)-stimulated ATPase, two well known plasma membrane enzyme markers. Uptake of nucleoside radioactivity was extensively studied in one subfraction; it was linear with time and membrane concentration over ranges used for the studies. Apparent Km values for uptake of radioactivity from adenosine, inosine, and uridine were 7.1 +/- 26 muM, respectively. Uptake of radioactivity from all three nucleosides exhibits a broad pH optimum from pH 7 to pH 9, but falls off rapidly at lower pH. N-Ethylmaleimide was an effective inhibitor of uptake of radioactivity from all three nucleosides; uptake of radioactivity from uridine is more sensitive than uptake of radioactivity from the purine nucleosides. Adenosine inhibited uptake of radioactivity from inosine more than from uridine. Inosine inhibited the uptake of radioactivity from adenosine, but uridine did not. Caffeine and 6-methylaminopurine riboside (6-N-methyladenosine differentially inhibit uptake of radioactivity from adenosine and inosine, and thus the vesicles apparently possess seperate transport systems for uptake of radioactivity from purine nucleosides and from uridine.     

Reconstitution studies of the human erythrocyte nucleoside transporter

The human erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 45,000-66,000) on the basis of reversible binding and photoaffinity labeling experiments with the nucleoside transport inhibitor, nitrobenzylthioinosine (NBMPR). In the present study, the NBMPR-binding protein was extracted from protein-depleted human erythrocyte "ghosts" with Triton X-100 and reconstituted into soybean phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes exhibited nitrobenzylthioguanosine (NBTGR)-sensitive [14C]uridine transport. A partially purified preparation of the NBMPR-binding protein, consisting largely of band 4.5 polypeptides, was also shown to have nucleoside transport activity. This band 4.5 preparation exhibited a 10-fold increase in uridine transport activity and a 7-fold increase in NBMPR-binding activity relative to the crude membrane extract. Uridine transport by the reconstituted band 4.5 preparation was saturable (apparent Km = 0.21 mM; Vmax = 9 nmol/mg of protein/5 s) and was inhibited by dipyridamole, dilazep, adenosine, and inosine. The vesicles reconstituted with the band 4.5 preparation also exhibited stereospecific glucose transport which was inhibited by cytochalasin B, but unaffected by NBTGR. In contrast, cytochalasin B was a poor inhibitor of NBTGR-sensitive uridine transport. These experiments implicate band 4.5 polypeptides in both nucleoside and sugar permeation.     

Detection and characterization of a nucleoside transport system in human fibroblast lysosomes

Lysosomes contain enzymatic activities capable of degrading nucleic acids to their constituent nucleosides, but the manner by which these degradation products are released from the lysosome is unknown. To investigate this process, human fibroblast lysosomes, purified on Percoll density gradients, were incubated with [3H]adenosine at pH 7.0, and the amount of adenosine taken up by the lysosomes was measured. Adenosine uptake by fibroblast lysosomes attained a steady state by 12 min at 37 degrees C and was unaffected by the presence of 2 mM MgATP or changes in pH from 5.0 to 8.0. An Arrhenius plot was linear with an activation energy of 12.9 kcal/mol and a Q10 of 2.0. Lysosomal adenosine uptake is saturable, displaying a Km of 9 mM at pH 7.0 and 37 degrees C. Various nucleosides and the nucleobase, 6-dimethylaminopurine, strongly inhibit lysosomal adenosine uptake, whereas neither D-ribose or nucleotide monophosphates have any significant effect upon lysosomal adenosine uptake. On a molar basis, purines are recognized more strongly than pyrimidines. Changing the nature of the nucleoside sugar from ribose to arabinose or deoxyribose has little effect on reactivity with this transport system. The known plasma membrane nucleoside transport inhibitors, dipyridamole and nitrobenzylthioinosine, inhibit lysosomal nucleoside transport at relatively low concentrations (25 microM) relative to the Km of 9 mM for lysosomal adenosine uptake. The half-times of [3H]inosine and [3H]uridine efflux from fibroblast lysosomes ranged from 6 to 8 min at 37 degrees C. Trans effects were not observed to be associated with either inosine or uridine exodus. In contrast to adenosine uptake, adenine primarily enters fibroblast lysosomes by a route not saturable by high concentrations of various nucleosides. In conclusion, the saturability of lysosomal adenosine uptake and its specific, competitive inhibition by other nucleosides indicate the existence of a carrier-mediated transport system for nucleosides within fibroblast lysosomal membranes.     

Nucleoside transport in rat cerebral-cortical synaptosomes. Evidence for two types of nucleoside transporters.

The transport of [U-14C]uridine was investigated in rat cerebral-cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions the rapid efflux of uridine from the synaptosomes is prevented and uridine is not significantly metabolized in the synaptosome during the first 1 min of uptake. The dose-response curve for the inhibition of uridine transport by nitrobenzylthioinosine (NBMPR) was biphasic: approx. 40% of the transport activity was inhibited with an IC50 (concentration causing half-maximal inhibition) value of 0.5 nM, but the remaining activity was insensitive to concentrations as high as 1 microM. Similar biphasic dose-response curves were observed for dilazep inhibition, but both transport components were equally sensitive to dipyridamole inhibition. Uridine influx by both components was saturable (Km 300 +/- 51 and 214 +/- 23 microM, and Vmax. 12 +/- 3 and 16 +/- 3 pmol/s per mg of protein, for NBMPR-sensitive and NBMPR-insensitive components respectively), and inhibited by other nucleosides such as 2-chloroadenosine, adenosine, inosine, thymidine and guanosine with similar IC50 values for the two components. Inhibition of uridine transport by NBMPR was associated with high-affinity binding of NBMPR to the synaptosome membrane (Kd 58 +/- 15 pM). Binding of NBMPR to these sites was competitively blocked by uridine and adenosine and inhibited by dilazep and dipyridamole, with Ki values similar to those measured for inhibiting NBMPR-sensitive uridine influx. These results demonstrate that there are two components of nucleoside transport in our rat synaptosomal preparation that differ in their sensitivity to inhibition by NBMPR. Thus conclusions regarding nucleoside transport in rat brain based only on NBMPR-binding activity must be viewed with caution.     

Two nucleoside uptake systems in Lactococcus lactis: competition between purine nucleosides and cytidine allows for modulation of intracellular nucleotide pools

A method for measuring internal nucleoside triphosphate pools of lactococci was optimized and validated. This method is based on extraction of (33)P-labeled nucleotides with formic acid and evaluation by two-dimensional chromatography with a phosphate buffer system for the first dimension and with an H(3)BO(3)-LiOH buffer for separation in the second dimension. We report here the sizes of the ribo- and deoxyribonucleotide pools in laboratory strain MG1363 during growth in a defined medium. We found that purine- and pyrimidine-requiring strains may be used to establish physiological conditions in batch fermentations with altered nucleotide pools and growth rates by addition of nucleosides in different combinations. Addition of cytidine together with inosine to a purine-requiring strain leads to a reduction in the internal purine nucleotide pools and a decreased growth rate. This effect was not seen if cytidine was replaced by uridine. A similar effect was observed if cytidine and inosine were added to a pyrimidine-requiring strain; the UTP pool size was significantly decreased, and the growth rate was reduced. To explain the observed inhibition, the nucleoside transport systems in Lactococcus lactis were investigated by measuring the uptake of radioactively labeled nucleosides. The K(m) for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition was found between uridine and either cytidine or inosine. These findings suggest that there are two different high-affinity nucleoside transporters, one system responsible for uridine uptake and another system responsible for the uptake of all purine nucleosides and cytidine.     

Investigation of Possible Participation of Nucleoside Transport Systems in the Postischemic Release of Purines and Pyrimidines from Cold Stored Liver

The aim of the study was to elucidate the role of nucleoside transport systems in the postischemic release of nucleosides and nucleobases accumulated by the rat liver during cold storage. Livers were preserved for 24 h in Euro-Collins (EC) or in a lactobionate-based solution (LBS) without exogenous adenosine. The rates of release of uric acid, xanthine, hypoxanthine, inosine, adenosine, uridine, and cytidine were monitored during early reperfusion. The greater part of the purines and pyrimidines (up to 80%) was lost in the first 2 min of reperfusion. After storage in EC, uric acid and xanthine formed more than 90% of the total purines released; nucleosides did not exceed 5% of the total. After storage in LBS, hypoxanthine formed more than 80% of purine efflux and the release of inosine and uridine was increased 5-10 times. These changes were shown to be due to the presence of allopurinol in LBS. Dipyridamole (an inhibitor of equilibrative nucleoside transporters) decreased the efflux of uric acid after storage in EC but residual release remained high. Dipyridamole exerted the most pronounced effect on the release of nucleosides (inosine and uridine) from livers stored in LBS. The use of sodium-free media for liver preservation and reperfusion did not alter the rates of purine and pyrimidine release. We conclude that equilibrative nucleoside transporters mediate the postischemic release of nucleosides and also, but to a less degree, of uric acid. Simple diffusion is an important factor in the release of nucleobases. Active Na(+)/nucleoside cotransport does not play an important role in early reperfusion.     

Transport of physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs by recombinant Escherichia coli nucleoside-H(+) cotransporter (NupC) produced in Xenopus laevis oocytes

The recently identified human and rodent plasma membrane proteins CNT1, CNT2 and CNT3 belong to a gene family (CNT) that also includes the bacterial nucleoside transport protein NupC. Heterologous expression in Xenopus oocytes has established that CNT1-3 correspond functionally to the three major concentrative nucleoside transport processes found in human and other mammalian cells (systems cit, cif and cib, respectively) and mediate Na(+) - linked uptake of both physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs. Here, one describes a complementary Xenopus oocyte transport study of Escherichia coli NupC using the plasmid vector pGEM-HE in which the coding region of NupC was flanked by 5'- and 3'-untranslated sequences from a Xenopus beta-globin gene. Recombinant NupC resembled human (h) and rat (r) CNT1 in nucleoside selectivity, including an ability to transport adenosine and the chemotherapeutic drugs 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxycytidine (ddC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine), but also interacted with inosine and 2',3'- dideoxyinosine (ddl). Apparent affinities were higher than for hCNT1, with apparent K(m) values of 1.5-6.3 microM for adenosine, uridine and gemcitabine, and 112 and 130 microM, respectively, for AZT and ddC. Unlike the relatively low translocation capacity of hCNT1 and rCNT1 for adenosine, NupC exhibited broadly similar apparent V(max) values for adenosine, uridine and nucleoside drugs. NupC did not require Na(+) for activity and was H(+) - dependent. The kinetics of uridine transport measured as a function of external pH were consistent with an ordered transport model in which H(+) binds to the transporter first followed by the nucleoside. These experiments establish the NupC-pGEM-HE/oocyte system as a useful tool for characterization of NupC-mediated transport of physiological nucleosides and clinically relevant nucleoside therapeutic drugs.     

A nucleoside transporter is functionally linked to ectonucleotidases in rat liver canalicular membrane.

Prevention of nucleoside loss in bile is physiologically desirable because hepatocytes are the main source of nucleosides for animal cells which lack de novo nucleoside biosynthesis. We have demonstrated a Na+ gradient-energized, concentrative nucleoside transport system in canalicular membrane vesicles (CMV) from rat liver by studying [3H]adenosine uptake using a rapid filtration technique. The Na(+)-dependent nucleoside transporter accepts purine, analogues of purine nucleosides and uridine; exhibits high affinity for adenosine (apparent Km, 14 microM); is not inhibited by nitrobenzylthioinosine or dipyridamole, and is present in CMV but not in rat liver sinusoidal membrane vesicles. Adenosine transport in right side-out CMV was substantially greater than with inside-out CMV. CMV also contain abundant ecto-ATPase and ecto-AMPase (5'-nucleotidase). These ectoenzymes were shown to degrade nucleotides into nucleosides which were conserved by the Na(+)-dependent nucleoside transport system.     

Independent blood-brain barrier transport systems for nucleic acid precursors.

The blood-brain barrier permeability to certain 14-C-labelled purine and pyrimidine compounds was studied by simultaneous injection in conjunction with two reference isotopes into the rat common carotid artery and decapitation 15s later. The amount of 14-C-labelled base or nucleoside remaining in brain was expressed in relation to 3-H2O (a highly diffusible internal standard) and 113m-In-labelled EDTA (an essentially non-diffusible internal standard). Of the 17 compounds tested, measurable, saturable uptakes were established for adenine, adenosine, guanosine, inosine and uridine. Two independent transport systems in the rat blood-brain barrier were defined. One transported adenine (Km equals 0.027 mM) and could be inhibited with hypoxanthine. Adenosine (Km equals 0.018 mM), guanosine, inosine and uridine all cross-inhibit, defining a second independent nucleoside carrier system. Adenosine inhibited [14-D]uridine uptake more effectively than did uridine, suggesting a weaker affinity of uridine for this nucleoside carrier.     

Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities

Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin B, the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a Km of 45 +/- 3 microM. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (IC50) observed at concentrations less than 30 microM. Of the pyrimidine nucleosides examined, only uridine (IC50, 41 +/- 9 microM) was a good inhibitor. Thymidine and cytidine were poor inhibitors with IC50 values greater than 300 microM. Direct measurements of [3H]thymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (IC50 values less than 25 microM), but not by formycin B, inosine, or guanosine (IC50 values greater than 600 microM). These data indicate that there are two sodium-dependent mechanisms for nucleoside transport in mouse intestinal epithelial cells, and that formycin B and thymidine may serve as model substrates to distinguish between these transporters. Neither of these sodium-dependent transport mechanisms was inhibited by nitrobenzylmercaptopurine riboside (10 microM), a potent inhibitor of one of the equilibrative (facilitated diffusion) nucleoside transporters found in many cells.     

Uptake and metabolism of purine nucleosides and nucleotides in isolated frog skeletal muscle.

When isolated frog skeletal muscles were incubated with ¹⁴C-labeled adenosine, the nucleoside was rapidly taken up by the cells and was either immediately incorporated into adenine nucleotides or deaminated to inosine. Incorporation was predominant at low (micromolar) concentrations whereas, deamination was the major route of metabolism at high (millimolar) concentrations. When muscles were incubated with ¹⁴C-labeled inosine the nucleoside, after entry into the cells, was metabolized to a lesser extent than adenosine. ATP and hypoxanthine were the major products of its metabolism. Intracellular concentrations were calculated using ³H-labeled sorbitol to measure the extracellular space.Because of its lower rate of intracellular metabolism inosine was used to investigate the characteristics of the nucleoside transport system. The uptake of inosine was saturable at high concentrations and was specifically inhibited by the presence of adenosine or uridine in the incubation media. Persantin, a well known specific inhibitor of nucleoside transport, also competitively inhibited inosine uptake, as did theophylline [1, Woo et al. Can J. Physiol. Pharmacol. $\text{52}$, 1063, 1974]. These data, along with the knowledge that in a well-oxygenated muscle, inosine entry follows a downhill chemical potential gradient, strongly support the view that the transport mechanism is facilitated diffusion.The muscle cell membrane does not appear to be permeable to ¹⁴C-labeled ATP under the conditions studied. Investigations of the permeability to the major extracellular degradation products of ATP suggest that AMP was the compound most likely to cross the cell membrane.     

Transport of nucleosides across the Plasmodium falciparum parasite plasma membrane has characteristics of PfENT1

Like all parasitic protozoa, the human malaria parasite Plasmodium falciparum lacks the enzymes required for de novo synthesis of purines and it is therefore reliant upon the salvage of these compounds from the external environment. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a nucleoside transporter that has been localized to the plasma membrane of the intraerythrocytic form of the parasite. In this study we have characterized the transport of purine and pyrimidine nucleosides across the plasma membrane of 'isolated' trophozoite-stage P. falciparum parasites and compared the transport characteristics of the parasite with those of PfENT1 expressed in Xenopus oocytes. The transport of nucleosides into the parasite: (i) was, in the case of adenosine, inosine and thymidine, very fast, equilibrating within a few seconds; (ii) was of low affinity [K(m) (adenosine) = 1.45 +/- 0.25 mM; K(m) (thymidine) = 1.11 +/- 0.09 mM]; and (iii) showed 'cross-competition' for adenosine, inosine and thymidine, but not cytidine. The kinetic characteristics of nucleoside transport in intact parasites matched very closely those of PfENT1 expressed in Xenopus oocytes [K(m) (adenosine) = 1.86 +/- 0.28 mM; K(m) (thymidine) = 1.33 +/- 0.17 mM]. Furthermore, PfENT1 transported adenosine, inosine and thymidine, with a cross-competition profile the same as that seen for isolated parasites. The data are consistent with PfENT1 serving as a major route for the uptake of nucleosides across the parasite plasma membrane.     

Functional production of mammalian concentrative nucleoside transporters in Saccharomyces cerevisiae

The transport of nucleosides and nucleobases in the yeast Saccharomyces cerevisiae is reviewed and the use of this organism to study recombinant mammalian concentrative nucleoside transport (CNT) proteins is described. A selection strategy based on the ability of an expressed nucleoside transporter cDNA to mediate thymidine uptake by yeast under a selective condition that depletes endogenous thymidylate was used to assess the transport capacity of heterologous transporter proteins. The pyrimidine-nucleoside selective concentrative transporters from human (hCNT1) and rat (rCNT1) complemented the imposed thymidylate depletion in S. cerevisiae, as did N-terminally truncated versions of hCNT1 and rCNT1 lacking up to 31 amino acids. Transporter-mediated rescue of S. cerevisiae by both nucleoside transporters was inhibited by cytidine, uridine and adenosine, but not by guanosine or inosine. This work represents the development of a new model system for the functional production of recombinant nucleoside transporters of the CNT family of membrane proteins.     

Characterization of inosine–uridine nucleoside hydrolase (RihC) from Escherichia coli

A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480 μM. No activity was exhibited against 2′-OH and 3′-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5′-OH group or with the 2′-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. I. Kinetics of incorporation of nucleosides into nucleotide pools and pool sizes during growth cycle

Results from kinetic studies on the incorporation of ³H-5-uridine and ³H-8-adenosine into the acid-soluble nucleotide poor and nucleic acids by Novikoff hepatoma cells (subline N1S1-67) in suspension culture indicate that the uridine transport reaction is saturated at about 100 μM and that for adenosine at about 10 μM nucleoside in the medium, and that above 100 μM simple diffusion becomes the predominant mode of entry of both nucleosides into the cell. The Km of the transport reactions is approximately 1.3 × 10^?5 M for uridine and 6 × 10^?6 M for adenosine. The incorporation of these nucleosides into both the nucleotide pool and into nucleic acids seems to be limited by the rate of entry of the nucleic acid synthesis from the rate of incorporation of nucleosides. Other complicating factors are a change with time of labeling in the relative proporation of nucleoside incorporated into DNA and into the individual nucleotides of RNA, the splitting of uridine to uracil by th ecells, the deamination of adenosine kto inosine and the subsequent cleavage of inosine to hypoxanthine. Various lines of evidence are presented which indicate that the overall nucleotide pools of the cells are very small under normal growth conditions. During growth in the presence of 200 μM uridine or adenosine, however, the cells continue to convert the nucleosides into intracellular nucleotides much more rapidly than required for nucleic acid synthesis. This results in an accumulation of free uridine and adenosine nucleotides in the cells, the maximum amounts of which are at least equivalent to the amount of these nucleotides in total cellular RNA.     

Functional production of mammalian concentrative nucleoside transporters in Saccharomyces cerevisiae

The transport of nucleosides and nucleobases in the yeast Saccharomyces cerevisiae is reviewed and the use of this organism to study recombinant mammalian concentrative nucleoside transport (CNT) proteins is described. A selection strategy based on the ability of an expressed nucleoside transporter cDNA to mediate thymidine uptake by yeast under a selective condition that depletes endogenous thymidylate was used to assess the transport capacity of heterologous transporter proteins. The pyrimidine-nucleoside selective concentrative transporters from human (hCNT1) and rat (rCNT1) complemented the imposed thymidylate depletion in S. cerevisiae, as did N-terminally truncated versions of hCNT1 and rCNT1 lacking up to 31 amino acids. Transporter-mediated rescue of S. cerevisiae by both nucleoside transporters was inhibited by cytidine, uridine and adenosine, but not by guanosine or inosine. This work represents the development of a new model system for the functional production of recombinant nucleoside transporters of the CNT family of membrane proteins.     

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.     

Sodium-dependent nucleoside transport in choroid plexus from rabbit. Evidence for a single transporter for purine and pyrimidine nucleosides.

The overall goal of this study was to determine the mechanisms by which nucleosides are transported in choroid plexus. Choroid plexus tissue slices obtained from rabbit brain were depleted of ATP with 2,4-dinitrophenol. Uridine and thymidine accumulated in the slices against a concentration gradient in the presence of an inwardly directed Na+ gradient. The Na(+)-driven uptake of uridine and thymidine was saturable with Km values of 18.1 +/- 2.0 and 13.0 +/- 2.3 microM and Vmax values of 5.5 +/- 0.3 and 1.0 +/- 0.2 nmol/g/s, respectively. Na(+)-driven uridine uptake was inhibited by naturally occurring ribo- and deoxyribonucleosides (adenosine, cytidine, and thymidine) but not by synthetic nucleoside analogs (dideoxyadenosine, dideoxycytidine, cytidine arabinoside, and 3'-azidothymidine). Both purine (guanosine, inosine, formycin B) and pyrimidine nucleosides (uridine and cytidine) were potent inhibitors of Na(+)-thymidine transport with IC50 values ranging between 5 and 23 microM. Formycin B competitively inhibited Na(+)-thymidine uptake and thymidine trans-stimulated formycin B uptake. These data suggest that both purine and pyrimidine nucleosides are substrates of the same system. The stoichiometric coupling ratios between Na+ and the nucleosides, guanosine, uridine, and thymidine, were 1.87 +/- 0.10, 1.99 +/- 0.35, and 2.07 +/- 0.09, respectively. The system differs from Na(+)-nucleoside co-transport systems in other tissues which are generally selective for either purine or pyrimidine nucleosides and which have stoichiometric ratios of 1. This study represents the first direct demonstration of a unique Na(+)-nucleoside co-transport system in choroid plexus.     

Sugar-free growth of mammalian cells on some ribonucleosides but not on others

It was shown earlier that a variety of vertebrate cells could grow indefinitely in sugar-free medium supplemented with either uridine or cytidine at greater than or equal to 1 mM. In contrast, most purine nucleosides do not support sugar-free growth for one of the following reasons. The generation of ribose-1-P from nucleoside phosphorylase activity is necessary to provide all essential functions of sugar metabolism. Some nucleosides, e.g. xanthosine, did not support growth because they are poor substrates for this enzyme. De novo pyrimidine synthesis was inhibited greater than 80% by adenosine or high concentrations of inosine, e.g. 10 mM, which prevented growth on these nucleosides; in contrast, pyrimidine synthesis was inhibited only marginally on 1 mM inosine or guanosine, but normal growth was only seen on 1 mM inosine, not on guanosine. The inhibition of de novo adenine nucleotide synthesis prevented growth on guanosine, since guanine nucleotides could not be converted to adenine nucleotides. Guanine nucleotides were necessary for this inhibition of purine synthesis, since a mutant blocked in their synthesis grew normally on guanosine. De novo purine synthesis was severely inhibited by adenosine, inosine, or guanosine, but in contrast to guanosine, adenosine and inosine could provide all purine requirements by direct nucleotide conversions.