Na(+)-dependent, active nucleoside transport in S49 mouse lymphoma cells and loss in AE-1 mutant deficient in facilitated nucleoside transport

S49 murine lymphoma cells were examined for expression of various nucleoside transport systems using a non-metabolized nucleoside, formycin B, as substrate. Nitrobenzylthioinosine (NBTI)-sensitive, facilitated transport was the primary nucleoside transport system of the cells. The cells also expressed very low levels of NBTI-resistant, facilitated nucleoside transport as well as of Na(+)-dependent, concentrative formycin B transport. Concentrative transport was specific for uridine and purine nucleosides, just as the concentrative nucleoside transporters of other mouse and rat cells. A nucleoside transport mutant of S49 cells, AE-1, lacked both the NBTI-sensitive, facilitated and Na(+)-dependent, concentrative formycin B transport activity, but Na(+)-dependent, concentrative transport of alpha-aminoisobutyrate was not affected.     

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.     

Sodium-dependent, concentrative nucleoside transport in Walker 256 rat carcinosarcoma cells

Nucleoside transport in Walker 256 cells was reexamined using formycin B, a nonmetabolized analog of inosine. In the presence of dipyridamole to inhibit the equilibrative (facilitated diffusion) transporter previously described in these cells, the initial rate of uptake of 1 microM formycin B was 10-fold greater in Na(+)-containing medium than in Na(+)-free medium. In the presence of Na+ and dipyridamole the intracellular concentration of formycin B exceeded that in the medium within one min and was 6-fold greater than that of the medium by 5 min. Na(+)-dependent transport of formycin B was inhibited by low concentrations of inosine, but not thymidine. Furthermore, Na(+)-dependent transport of uridine, but not thymidine, was apparent in the presence of dipyridamole. These data indicate that Walker 256 cells have, in addition to the previously described equilibrative transporter, a concentrative nucleoside transporter. The specificity of this transporter appears to correspond to one of the two Na(+)-dependent transporters previously described in mouse intestinal epithelial cells.     

Sodium-dependent, concentrative nucleoside transport in cultured intestinal epithelial cells

In a simple salts medium, monolayers of IEC-6 intestinal cells achieved concentrations of unmetabolized formycin B (an analog of inosine) about 6-fold higher than in the medium. Rates of formycin B influx were a saturable function of Na+ concentrations in the medium. Although IEC-6 cells possess sites with high affinity for nitrobenzylthioinosine, a potent inhibitor of equilibrative (facilitated diffusion) nucleoside transport systems in certain cell types, the inhibitor had only minor effects on formycin B uptake in IEC-6 cells, but reduced efflux of the analog from these cells. These findings indicate the joint presence in IEC-6 cells of nucleoside transporters of two types, one that is concentrative and Na+-dependent, and another that is sensitive to nitrobenzylthioinosine and apparently equilibrative.     

Characterization of Na(+)-dependent, active nucleoside transport in rat and mouse peritoneal macrophages, a mouse macrophage cell line and normal rat kidney cells

Peritoneal rat macrophages expressed solely an Na(+)-dependent, concentrative nucleoside transporter, which possesses a single Na(+)-binding site and transports purine nucleosides and uridine but not thymidine or deoxycytidine. The Michaelis-Menten constants for formycin B and Na+ were about 6 microns and 14 mM, respectively, and the estimated Na+:formycin B stoichiometry was 1:1. Rat macrophages accumulated 5 microM formycin B to a steady-state level exceeding that in the medium by about 500-fold during 60 min of incubation at 37 degrees C. Concentrative formycin B transport was resistant to inhibition by nitrobenzylthioinosine, lidoflazine, dilazep and nifedipine, but was slightly inhibited by high concentrations of dipyridamole (greater than 10 microM) and probenecid (greater than 100 microM). Mouse peritoneal macrophages and lines of mouse macrophages and normal rat kidney cells expressed Na(+)-dependent, active nucleoside transport but in addition significant Na(+)-independent, facilitated nucleoside transport. Facilitated nucleoside transport in these cells was sensitive to inhibition by nitrobenzylthioinosine, dilazep and dipyridamole. The presence of these inhibitors greatly enhanced the concentrative accumulation of formycin B by these cells by inhibiting the efflux via the facilitated transporter of the formycin B actively transported into the cells. Whereas rat macrophages lacked high-affinity nitrobenzylthioinosine-binding sites, mouse macrophages and normal rat kidney cells possessed about 10,000 such sites/cell. Rat and mouse erythrocytes, rat lymphocytes, and lines of Novikoff rat hepatoma cells, Chinese hamster ovary cells, Mus dunni cells and embryonic monkey kidney cells expressed only facilitated nucleoside transport.     

Na(+)-dependent, active nucleoside transport in mouse spleen lymphocytes, leukemia cells, fibroblasts and macrophages, but not in equivalent human or pig cells; dipyridamole enhances nucleoside salvage by cells with both active and facilitated transport

Formycin B influx studies have shown that P388 and L1210 mouse leukemia cells, mouse L929 cells, mouse RAW 309 Cr.1 cells, LK35.2 mouse B-cell hybridoma cells and cultured mouse peritoneal macrophages express both Na(+)-dependent, active and nonconcentrative, facilitated nucleoside transport systems. In the mouse cell lines, active transport represented only a minor nucleoside transport component and was detected only by measuring formycin B uptake in the presence of dipyridamole or nitrobenzylthioinosine, strong inhibitors of facilitated, but not of active, nucleoside transport. Inhibition of facilitated transport resulted in the concentrative accumulation of formycin B in cells expressing active nucleoside transport. Concentrative formycin B accumulation was abolished by treatment of the cells with gramicidin or absence of Na+ in the extracellular medium and strongly inhibited by ATP depletion or ouabain treatment. Mouse macrophages accumulated formycin B to 70-times the extracellular concentration in the absence of dipyridamole during 90 min of incubation at 37 degrees C. Thus active transport represents a major nucleoside transport system of these cells, similarly as previously reported for mouse spleen lymphocytes. In contrast to the various types of mouse cells, active formycin B transport was not detected in human HeLa cells, human H9, Jurkat and CEM T lymphoidal cells and pig spleen lymphocytes. These cells expressed only facilitated nucleoside transport with kinetic properties similar to those of the facilitated transporters of other mammalian cells.     

Characterization of equilibrative and concentrative Na+-dependent (cif) nucleoside transport in acute promyelocytic leukemia NB4 cells

Nucleoside transport processes can be classified by the transport mechanism, e = equilibrative and c = concentrative, by the sensitivity to inhibition by nitrobenzylthioinosine (NBMPR), s = sensitive and i = insensitive, and also by permeant selectivity. To characterize nucleoside transport in acute promyelocytic NB4 cells, nucleoside transport was resolved into different components by selective elimination of transport processes with NBMPR and with Na⁺-deficient media. Initial transport rates were estimated from time course experiments. For adenosine, uridine, and formycin B, equilibrative transport accounted for approximately 60% of their uptake, with ei and es transport contributing almost equally, and Na⁺-dependent transport accounting for the remaining 40% of the total uptake. Thymidine uptake was mediated exclusively by equilibrative systems with ei and es systems each contributing 50% to total uptake. Adenosine accumulated above equilibrative concentrations, suggesting that a concentrative transport process was active and/or that metabolism led to adenosine's accumulation. Formycin B, a nonmetabolizable analog, also accumulated in the cells, supporting the concentrative potential of the Na⁺-dependent transporter. Kinetic analyses also provided evidence for three distinct high affinity transport mechanisms. NBMPR binding assays indicated the presence of two high affinity (K_m 0.10 and 0.35 nM) binding sites. In conclusion, NB4 cells express ei and es transport, as well as a large ci transport component, which appears to correspond to cif (f = formycin B or purine selective) nucleoside transport, not previously described in human cells. © 1996 Wiley-Liss, Inc.     

Sodium-dependent nucleoside transport in mouse leukemia L1210 cells

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.     

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.     

Recent advances in the molecular biology of nucleoside transporters of mammalian cells.

Nucleosides are hydrophilic molecules and require specialized transport proteins for permeation of cell membranes. There are two types of nucleoside transport processes: equilibrative bidirectional processes driven by chemical gradients and inwardly directed concentrative processes driven by the sodium electrochemical gradient. The equilibrative nucleoside transport processes (es, ei) are found in most mammalian cell types, whereas the concentrative nucleoside transport processes (cit, cif, cib, csg, cs) are present primarily in specialized epithelia. Using a variety of cloning strategies and functional expression in oocytes of Xenopus laevis, we have isolated and characterized cDNAs encoding the rat and human nucleoside transporter proteins of the four major nucleoside transport processes of mammalian cells (es, ei, cit, cif). From the sequence relationships of these proteins with each other and with sequences in the public data bases, we have concluded that the equilibrative and concentrative nucleoside transport processes are mediated by members of two previously unrecognized groups of integral membrane proteins, which we have designated the equilibrative nucleoside transporter (ENT) and the concentrative nucleoside transporter (CNT) protein families. This review summarizes the current state of knowledge in the molecular biology of the ENT and CNT protein families, focusing on the characteristics of the four human (h) and rat (r) nucleoside transport proteins (r/hENT1, r/hENT2, r/hCNT1, r/hCNT2).     

Isolation and characterization of an L1210 cell line retaining the sodium-dependent carrier cif as its sole nucleoside transport activity

Nucleoside permeation across mammalian cell membranes is complex with at least four distinct transporters known. Two of these (es and ei) are equilibrative (facilitated diffusion) carriers that have been studied is considerable detail. The other two (cif and cit) are concentrative, Na(+)-dependent carriers. A major obstacle to the characterization of the latter two mechanisms has been the lack of suitable model systems expressing only a single nucleoside transport activity. The present study describes the isolation of a cell line that has cif as its sole nucleoside transporter. L1210/MC5-1 cells, which have es and cif transport activity, were mutagenized and plated in soft agar containing two cytotoxic nucleosides (tubercidin (7-deazaadenosine) and cytosine arabinoside) that are substrates for es but not cif. A clonal line (L1210/MA-27.1) was isolated which retained the capacity for Na(+)-dependent [3H]formycin B transport but was unable to transport [3H]thymidine, a substrate for es but not cif. Failure of the mutant to transport thymidine was also demonstrated by the inability of thymidine (with adenine as a purine source) to rescue these cells from methotrexate toxicity. Furthermore, the mutant lacked nitrobenzylthioinosine (NBMPR) binding activity (an integral part of the es transporter) as demonstrated by reversible NBMPR binding and photoaffinity labeling with [3H]NBMPR. Loss of es transport activity was also demonstrated by the failure of NBMPR to affect the toxicity of 2-chlorodeoxyadenosine (IC50 approximately 30 nM) in L1210/MA27.1 cells. In contrast, NBMPR decreased the IC50 for 2-chlorodeoxyadenosine from 100 to 30 nM in the parental L1210/MC5-1 cell line. These results are consistent with the mechanism of NBMPR potentiation of 2-chlorodeoxyadenosine toxicity in L1210 cells being a blockade of efflux via es while the nucleoside is pumped into the cells by the concentrative cif carrier.     

L1210/B23.1 cells express equilibrative, inhibitor-sensitive nucleoside transport activity and lack two parental nucleoside transport activities.

Cultured mouse leukemia L1210 cells express the nucleoside-specific membrane transport processes designated es, ei, and cif. The es and ei processes are equilibrative, but may be distinguished by the high sensitivity of the former to 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR); the cif process is mediated by a Na+/nucleoside cotransporter of low sensitivity to NBMPR. Cells of an ei-deficient clonal line, L1210/MC5-1, were mutagenized, and clones were selected in soft agar medium that contained (i) NBMPR (an inhibitor of es processes), (ii) erythro-9-(2-hydorxy-3-nonyl)adenine (an inhibitor of adenosine deaminase), and (iii) arabinofuranosyladenine (a cytotoxic substrate for the three nucleotide transporters). The selection medium did not allow es activity and selected against cells that expressed the Na(+)-linked cif process. Cells of the L1210/B23.1 clonal isolate were deficient in cif transport activity, and inward fluxes of formycin B, a poorly metabolized analog of inosine, were virtually abolished by NBMPR in these cells. In the mutant cells, nonisotopic formycin B behaved as a countertransport substrate during influx of [3H]formycin B, and inward fluxes of the latter were competitively inhibited by purine and pyrimidine nucleosides. The transport behavior of L1210/B23.1 cells indicates that (i) the mutation/selection procedure impaired or deleted the Na(+)-linked cif process and (ii) es nucleoside transport activity is expressed in the mutant cells.     

Nucleoside transport in L1210 murine leukemia cells. Evidence for three transporters

L1210 murine leukemia cells have two nucleoside transport activities that differ in their sensitivity to nitrobenzylmercaptopurine riboside (NBMPR). This study re-examines NBMPR-insensitive nucleoside transport in these cells and finds that it is mediated by two components, one Na(+)-dependent and the other Na(+)-independent. A mutant selected previously for loss of NBMPR-insensitive transport lacks only the Na(+)-independent activity. When NBMPR is used to block efflux via the NBMPR-sensitive transporter, uptake of formycin B (a nonmetabolized analog of inosine) is concentrative in both the parental and mutant cells, but the intracellular concentration of the nucleoside is 5-fold lower in the parental cells. Decreased accumulation of formycin B in the parental cells is due to efflux of the nucleoside via the NBMPR-insensitive, Na(+)-independent transporter that the mutant lacks. The Na(+)-dependent transporter appears to accept most purine, but not pyrimidine, nucleosides as substrates. Two exceptions are uridine, a good substrate, and 7-deazaadenosine, a poor substrate. In contrast, all of the nucleosides tested are substrates for the Na(+)-independent transporter. We conclude that L1210 cells have three distinct nucleoside transporters and that the specificity of the Na(+)-dependent transporter is similar to that of one of the two Na(+)-dependent nucleoside transporters seen in mouse intestinal epithelial cells.     

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.     

The effect of formycin B on mRNA translation and uptake of purine precursors in Leishmania mexicana

Formycin B is a structural analog of inosine that is a potent inhibitor of Leishmania multiplication. Formycin B is reportedly converted to formycin A nucleotides and incorporated into RNA by the organisms, and it is unclear whether the active form of the drug is the nucleoside itself or its several metabolites. We confirmed that formycin A nucleotides are formed by formycin B-exposed L. mexicana promastigotes, and determined that the intraparasite concentration of Formycin B and its metabolites was 6 times the extracellular formycin B concentration. Formycin B did not significantly inhibit purine nucleoside transport by intact promastigotes or purine base phosphoribosylation by parasite lysates. Thus, the nucleoside does not appear to inhibit these initial steps of purine nucleoside metabolism. Since RNA and protein synthesis in formycin B-treated intact promastigotes was found to be inhibited within 30 minutes, the effect of formycin A metabolites on leishmanial protein synthesis was investigated in in vitro protein synthesis experiments. Messenger RNA from formycin B-treated promastigotes was translated only 40% as efficiently as control promastigote mRNA by rabbit reticulocyte lysates. In addition, when formycin A-5'-triphosphate was preincubated with the rabbit reticulocyte lysates, translation of control mRNA was 86% inhibited. Formycin B toxicity to Leishmania promastigotes appears to be at least partially due to inhibition of protein synthesis by formycin A nucleotides and formycin A containing mRNA.     

Macrophages require different nucleoside transport systems for proliferation and activation.

To evaluate the mechanisms involved in macrophage proliferation and activation, we studied the regulation of the nucleoside transport systems. In murine bone marrow-derived macrophages, the nucleosides required for DNA and RNA synthesis are recruited from the extracellular medium. M-CSF induced macrophage proliferation and DNA and RNA synthesis, whereas interferon gamma (IFN-gamma) led to activation, blocked proliferation, and induced only RNA synthesis. Macrophages express at least the concentrative systems N1 and N2 (CNT2 and CNT1 genes, respectively) and the equilibrative systems es and ei (ENT1 and ENT2 genes, respectively). Incubation with M-CSF only up-regulated the equilibrative system es. Inhibition of this transport system blocked M-CSF-dependent proliferation. Treatment with IFN-gamma only induced the concentrative N1 and N2 systems. IFN-gamma also down-regulated the increased expression of the es equilibrative system induced by M-CSF. Thus, macrophage proliferation and activation require selective regulation of nucleoside transporters and may respond to specific requirements for DNA and RNA synthesis. This report also shows that the nucleoside transporters are critical for macrophage proliferation and activation.     

Long term endocrine regulation of nucleoside transporters in rat intestinal epithelial cells

We studied the regulation of nucleoside transporters in intestinal epithelial cells upon exposure to either differentiating or proliferative agents. Rat intestinal epithelial cells (line IEC-6) were incubated in the presence of differentiating (glucocorticoids) or proliferative (EGF and TGF-alpha) agents. Nucleoside uptake rates and nucleoside transporter protein and mRNA levels were assessed. The signal transduction pathways used by the proliferative stimuli were analyzed. We found that glucocorticoids induce an increase in sodium-dependent, concentrative nucleoside transport rates and in protein and mRNA levels of both rCNT2 and rCNT1, with negligible effects on the equilibrative transporters. EGF and TGF-alpha induce an increase in the equilibrative transport rate, mostly accounted for by an increase in rENT1 activity and mRNA levels, rENT2 mRNA levels remaining unaltered. This effect is mimicked by another proliferative stimulus that functions as an in vitro model of epithelial wounding. Here, rENT1 activity and mRNA levels are also increased, although the signal transduction pathways used by the two stimuli are different. We concluded that differentiation of rat intestinal epithelial cells is accompanied by increased mature enterocyte features, such as concentrative nucleoside transport (located at the brush border membrane of the enterocyte), thus preparing the cell for its ultimate absorptive function. A proliferative stimulus induces the equilibrative nucleoside activities (mostly through ENT1) known to be located at the basolateral membrane, allowing the uptake of nucleosides from the bloodstream for the increased demands of the proliferating cell.     

Nucleoside transport in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. Differences in sensitivity to nitrobenzylthioinosine and thiol reagents.

The characteristics of nucleoside transport were examined in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. In Walker 256 cells the initial rates of uridine, thymidine and adenosine uptake were insensitive to the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR) (1 microM), but were partially inhibited by dipyridamole (10 microM), another inhibitor of nucleoside transport. In contrast, the transport of these nucleosides in S49 cells was completely blocked by both inhibitors. Nucleoside transport in Walker 256 and S49 cells also differed in its sensitivity to the thiol reagent p-chloromercuribenzenesulphonate (pCMBS). Uridine transport in Walker 256 cells was inhibited by pCMBS with an IC50 (concentration producing 50% inhibition) of less than 25 microM, and inhibition was readily reversed by beta-mercaptoethanol. In S49 cells uridine transport was only inhibited at much higher concentrations of pCMBS (IC50 approximately equal to 300 microM). In other respects nucleoside transport in Walker 256 and S49 cells were quite similar. The Km and Vmax. values for uridine transport were nearly identical, and the transporters of both cell lines appeared to accept a broad range of nucleosides as substrates. Uridine transport in Walker 256 cells was non-concentrative and did not require an energy source. These studies demonstrate that nucleoside uptake in Walker 256 cells is mediated by a facilitated-diffusion mechanism which differs markedly from that of S49 cells in its sensitivity to the transport inhibitor NBMPR and the thiol reagent pCMBS.     

Nucleoside transport in human colonic epithelial cell lines: evidence for two Na+-independent transport systems in T84 and Caco-2 cells.

RT-PCR of RNA isolated from monolayers of the human colonic epithelial cell lines T84 and Caco-2 demonstrated the presence of mRNA for the two cloned Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2, but not for the cloned Na+-dependent concentrative nucleoside transporters, CNT1 and CNT2. Uptake of [3H]uridine by cell monolayers in balanced Na+-containing and Na+-free media confirmed the presence of only Na+-independent nucleoside transport mechanisms. This uptake was decreased by 70-75% in the presence of 1 microM nitrobenzylthioinosine, a concentration that completely inhibits ENT1, and was completely blocked by the addition of 10 microM dipyridamole, a concentration that inhibits both ENT1 and ENT2. These findings indicate the presence in T84 and Caco-2 cells of two functional Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2.     

Characterization of nucleoside transport systems in cultured rat epididymal epithelium

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na(+) dependent, while 30% was Na(+) independent. The Na(+)-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na(+)-dependent [(3)H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na(+)-dependent [(3)H]guanosine uptake was inhibited by thymidine by 20% and Na(+)-dependent [(3)H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na(+)-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.