Enhancement of retention and cytotoxicity of 2-chlorodeoxyadenosine in cultured human leukemic lymphoblasts by nitrobenzylthioinosine, an inhibitor of equilibrative nucleoside transport.

In leukemic cells exposed to 2-chlorodeoxyadenosine (2-CdA), levels of the nucleoside drug and its phosphate metabolites decay with time in the absence of external 2-CdA; an intrinsic part of this process is the efflux of 2-CdA. The effects of nitrobenzylthioinosine (NBMPR) and of dipyridamole (DPM), both potent inhibitors of es (e, equilibrative; s, sensitive to NBMPR) nucleoside transport processes, were studied in four lines of cultured leukemic lymphoblasts. Suspensions of 2-CdA-loaded cells were diluted 10-fold with 2-CdA-free medium to initiate the cellular 2-CdA decay processes, which followed a biexponential time course. When diluting media contained NBMPR or DPM, intracellular levels of 2-CdA and its metabolites were substantially increased (P < 0.001) compared with cells in media lacking the transport inhibitors, and 2-CdA loss followed a monoexponential time course. As a consequence, the AUCs (area under time-course plots of intracellular 2-CdA and its metabolites) were significantly (P < 0.001) lower in untreated control cells compared to inhibitor-treated cells. These results suggest that nucleoside transport processes contribute to the efflux of 2-CdA from the cultured lymphoblasts. The cytotoxicity of 1-h exposure to 2-CdA of Reh-A2 and CCRF-CEM cells was enhanced three-fold by subsequent exposure to 0.5 microM NBMPR relative to that of control cells subjected to the same manipulations without NBMPR exposure. However, before such a strategy may be considered to have a therapeutic application, careful examination of effects in normal lymphocytes and ex vivo leukemic lymphoblasts must first be undertaken. Leukemia (2000) 14, 52-60.

Es nucleoside transporter content of acute leukemia cells: role in cell sensitivity to cytarabine (araC).

Nucleoside analogs are important components of treatment regimens for acute leukemia in adults. Plasma membrane permeation of the nucleoside analog molecules, the initial event in the cellular conversion of nucleosides to active agents, is mediated by nucleoside-specific membrane transporters. The widely-expressed es nucleoside transporter accepts as substrates diverse nucleoside analogs, including cytarabine (araC), 2-chlorodeoxyadenosine, and fludarabine. The cellular content of es transporter sites has been measured in blasts from patients with acute lymphoblastic leukemia and acute myelogenous leukemia, by a sensitive, quantitative flow cytometry assay that employs the tightly-bound es ligand, SAENTA fluorescein. Values for es transporter expression varied ten-fold among samples from patients with acute myelogenous leukemia. In this article, we review current findings that document, in confocal fluorescence microscopy images and in flow cytometry assays of SAENTA fluorescein-stained cells, the patient-to-patient variance of es transporter expression in leukemic blasts from patients. Our data show a correlation between the expression of es transporters and the in vitro sensitivity to nucleoside drugs of blasts from acute leukemia patients. These findings show that the flow cytometry assay of es expression provides a facile means of predicting resistance of leukemia cells to the cytotoxicity of araC and other nucleosides.

Sensitivity of acute leukemia cells to cytarabine is a correlate of cellular es nucleoside transporter site content measured by flow cytometry with SAENTA-fluorescein.

Cytarabine (araC) is converted to araC 5'-triphosphate after entering leukemia cells as a substrate for nucleoside transport processes. This study tested the relationship between araC cytotoxicity, measured in an in vitro tetrazolium dye reduction assay of cell viability, and the cellular abundance of es nucleoside transport elements, assayed by a flow cytometric method that used the es-specific stain, 5-(SAENTA-x8)-fluorescein (5-(Sx8)-F), in cultured leukemia cells and in myeloblasts and lymphoblasts (blasts) from leukemia patients. Cellular es site abundance (B(max) value for 5-(Sx8)-F binding) varied sixfold among nine leukemic myeloblast samples from patients. In cultured OCI/AML-2 myeloblasts and CCRF-CEM T-lymphoblasts, and in fresh leukemic blasts, es sites were fractionally blocked by treatment with graded concentrations of nitrobenzylthioinosine (NBMPR), an inhibitory es site ligand, to simulate the variation in es expression found in leukemic blasts from patients with acute myeloid leukemia. When the cytotoxicity of a single concentration of araC was determined in NBMPR-treated leukemia cells, cell kill correlated closely with the intensity of 5-(Sx8)-F fluorescence (r = .92 to .99), a measure of the cell surface abundance of functional es nucleoside transporter sites. Concentrations of NBMPR that achieved half-maximal reduction (4.3 to 12 nmol/L) of cellular 5-(Sx8)-F fluorescence (measured by flow cytometry) approximated IC50 values (1 to 10 nmol/L) previously found for inhibition by NBMPR of es-mediated nucleoside fluxes in several cell types, supporting the view that 5-(Sx8)-F interacted with the estransporter. The correlation of araC cytotoxicity and the B(max) for 5-(Sx8)-F binding to es sites in cultured leukemia cells and in leukemic blasts from acute leukemia patients (r = .95) suggests that the flow cytometry assay of es capacity may be useful in predicting clinical response to araC.

Modulation of the metabolism of beta-L-(-)-2',3'-dideoxy-3'-thiacytidine by thymidine, fludarabine, and nitrobenzylthioinosine.

beta-L-(-)-2',3'-Dideoxy-3'-thiacytidine (3TC) is a cytosine nucleoside analog that potently inhibits the replication of human and duck hepatitis B viruses and human immunodeficiency virus through the activity of its 5'-triphosphate ester metabolite. The present study examined the intracellular decay of 3TC 5'-phosphates and tested strategies for modulating the cellular content of those nucleotides in primary cultures of duck hepatocytes and in human hepatoma 2.2.15 cells and CCRF-CEM T lymphoblasts. Inhibition by deoxycytidine of the 5'-phosphorylation of 3TC in duck hepatocytes confirmed that, as in mammalian cells, deoxycytidine kinase catalyzed 3TC activation. The 5'-mono, 5'-di-, and 5'-triphosphates of 3TC underwent monoexponential elimination from duck hepatocytes and 2.2.15 cells (half-lives, 3.6 to 8.0 h). Thymidine and fludarabine, which are agents that enhance the activity of deoxycytidine kinase, were tested in strategies for increasing the cellular content of 3TC 5'-phosphates. Coordinate treatment of cells with 3TC and thymidine (50 microM) increased the content of 3TC 5'-monophosphate in duck hepatocytes and the content of 3TC 5'-di- and 5'-triphosphates in 2.2.15 cells, but enhancement of 3TC 5'-phosphate levels in CCRF-CEM cells required a higher thymidine concentration (100 microM). Fludarabine (5 microM) did not affect the contents of 3TC 5'-di- and 5'-triphosphates in duck hepatocytes, but modestly increased the contents of those nucleotides in 2.2.15 cells and CCRF-CEM cells. Nitrobenzylthioinosine (NBMPR), an inhibitor of the es facilitated diffusion nucleoside transporter, reduced the level of entry of 3TC into 2.2.15 cells and abolished inward fluxes of thymidine, adenosine, and deoxycytidine. In 2.2.15 cells and CCRF-CEM cells, NBMPR reduced the formation of 3TC 5'-di- and 5'-triphosphates and reversed the thymidine- and fludarabine-induced increases in the formation of those nucleotides. NBMPR protected against the cytotoxicity of 3TC in CCRF-CEM cells, whereas thymidine potentiated that toxicity, apparently by enhancing the formation of 3TC 5'-triphosphate. Taken together, these results indicate that deoxycytidine kinase and the es nucleoside transporter are targets for manipulation of the metabolism and activity of 3TC.

Measurement of nitrobenzylthioinosine in plasma and erythrocytes: a pharmacokinetic study in mice.

PURPOSE: Nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport in many cell types, modulates the in vivo disposition of several cytotoxic nucleoside analogs. In this study, a radioligand binding assay was developed for measurement of the NBMPR content of plasma and erythrocytes. METHODS: The assay was based on the competition between NBMPR and [3H]NBMPR for high-affinity sites on human erythrocytes membranes. With this assay, we followed in mice changes in the NBMPR content of blood plasma and erythrocytes, following the intraperitoneal injection of the disodium salt of NBMPR 5'-monophosphate (NBMPR-P), a prodrug form of NBMPR. RESULTS: The radioligand binding assay was able to measure precisely as little as 2.5 pmol of NBMPR, allowing the direct determination of NBMPR concentrations in plasma as low as 16 nM. As few as 8 x 10(3) molecules of NBMPR per cell could be determined in erythrocytes. The NBMPR content of plasma from mice injected with NBMPR-P was maximal at about 20 min after injection and declined to < 0.2% of the peak value by 10 h. Erythrocyte-associated NBMPR was also maximal at 20 min, and declined to 11% of the peak value by 10 h after injection. Time courses for the disappearance of NBMPR from plasma and erythrocytes were monoexponential and yielded half-life values of 0.39 h and 0.68 h, respectively, an apparent volume of distribution of 0.61 l/kg, and a clearance of 1.1 l/h per kg. CONCLUSIONS: The radioligand binding assay is a sensitive and facile method for monitoring NBMPR concentrations in mammalian plasma and tissue extracts.

Transport of adenosine by recombinant purine- and pyrimidine-selective sodium/nucleoside cotransporters from rat jejunum expressed in Xenopus laevis oocytes.

Two major Na+-dependent nucleoside transporter subtypes implicated in adenosine transport in mammalian cells are distinguished functionally on the basis of substrate specificity: one is selective for pyrimidine nucleosides but also binds adenosine, and the other has selectivity for purine nucleosides but also binds uridine. Transportability of adenosine by the purine-selective system has been established by measurements of [3H]adenosine fluxes, whereas the conclusion that adenosine is permeant of the pyrimidine-selective system is based on inhibition assays. We investigated adenosine transport mediated by a recombinant pyrimidine-selective rat jejunal/kidney Na+/nucleoside cotransporter (rCNT1) expressed in Xenopus laevis oocytes and compared it with that mediated by a recombinant purine-selective rat jejunal/liver Na+/nucleoside cotransporter (rCNT2). Adenosine fluxes mediated by rCNT1 were 1 order of magnitude lower than those mediated by rCNT2. In kinetic studies, rCNT1 transported adenosine with an apparent Km value for influx (26 microM) similar to that for uridine but with a very much lower Vmax value, and the Vmax/Km ratios were 0.003 and 0.57 for adenosine and uridine, respectively. Recombinant rCNT1 mediated efflux of [3H]uridine from preloaded oocytes, demonstrating a capacity for bidirectional transport of nucleoside permeants. Uridine efflux was stimulated by extracellular uridine and inhibited by extracellular adenosine, suggesting that the rate of conversion of rCNT1 from its outward-facing conformation to its inward-facing conformation was increased when the transporter was complexed with uridine and decreased when it was complexed with adenosine. Thus, although rCNT1 binds adenosine and uridine with similar affinities, it kinetically favors transport of uridine.

Intracellular pharmacokinetics of 2-chlorodeoxyadenosine in leukemia cells from patients with chronic lymphocytic leukemia.

2-Chlorodeoxyadenosine (2-CdA) is an important agent in the treatment of hairy cell leukemia and chronic lymphocytic leukemia (CLL). Others have reported that levels of 2-CdA phosphates present in human leukemia cells decline rapidly when the cells are in 2-CdA-free medium (Santana et al. J Clin Oncol 1991; 9: 416-422). In the present study, time-courses of 2-CdA loss from CLL cells were biexponential: the mean half-life of the initial phase was 0.30 +/- 0.18 h; the presence of 0.5 microM nitrobenzylthioinosine (NBMPR, a classical inhibitor of nucleoside transport) in the suspending medium, significantly decreased the initial rate of 2-CdA efflux (mean half-life, 0.43 +/- 0.22 h). As a consequence, AUCs (areas under time-course plots) were significantly higher in the NBMPR-treated cells (4.56 +/- 2.01 pmol.h/10(6) cells, n = 19) than in untreated control cells (3.83 +/- 1.74 pmol.h/10(6) cells; n = 19). 2-CdA was the principal efflux product released into the medium from 2-CdA-loaded CLL cells. We conclude that nucleoside transport processes contribute to the efflux of 2-CdA from CLL cells and that NBMPR may be useful as a retentive agent.

Nucleobase transporter-mediated permeation of 2',3'-dideoxyguanosine in human erythrocytes and human T-lymphoblastoid CCRF-CEM cells.

Several 2',3'-dideoxynucleosides (ddNs), agents that inhibit the replication of human immunodeficiency virus and hepatitis B virus, enter mammalian cells by simple diffusion. In this report, we show that the membrane permeation of 2',3'-dideoxyguanosine (ddG) in human erythrocytes and CCRF-CEM cells, in contrast with that of other ddNs, is transporter-mediated. Inward fluxes of ddG in both cell types were inhibited by adenine, hypoxanthine, and acyclovir, but not by inhibitors of nucleoside transport (nitrobenzylthioinosine, dipyridamole, dilazep). Fluxes of ddG in human erythrocytes were attributable to a single, rate-saturable process (Km, 380 +/- 90 microM and Vmax, 7.9 +/- 0.8 pmol/s/microliter cell water) that was competitively inhibited by adenine (Ki, 16 microM). These results showed that ddG entered human erythrocytes and CCRF-CEM cells by a transporter-mediated process that was also the basis for entry of purine nucleobases. In contrast, inward fluxes of 2,6-diaminopurine-2',3'-dideoxyriboside (ddDAPR), a prodrug of ddG, were not affected by purine nucleobases or nucleoside transport inhibitors in either cell type. Thus, the permeation properties of ddDAPR resembled those of 2',3'-dideoxyadenosine, a diffusional permeant (cell uptake is transporter-independent), and contrasted with those of ddG, the deamination product of ddDAPR. This study demonstrated that the nucleobase moiety of ddNs is an important determinant of membrane permeation.

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.

Synthesis and biological activity of 5-(2,2-difluorocyclopropyl)-2'-deoxyuridine deoxyuridine diastereomers.

The synthesis of the two diastereomers (9 and 10) of 5-(2,2-difluorocyclopropyl)-2'-deoxyuridine are described. Their antiviral and cytotoxic activities were determined, in comparison with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 5-fluoro-2'-deoxyuridine (FDU), respectively. 5-[(1R)-2,2-Difluorocyclopropyl]-2'-deoxyuridine (10) was the most active antiviral agent against HSV-1 (IC50 = 5 micrograms/ml) relative to BVDU (IC50 = 0.082 micrograms/ml), and cytotoxic agent in the CCRF-CEM (IC50 = 230 microM) screen relative to FDU (IC50 = 4.7 x 10(-3) microM). The 5-[(1S)-2,2-difluorocyclopropyl] diastereomer was inactive in both screens. Partition coefficients (P) and affinity for the mouse erythrocyte nucleoside transporter (Ki) were not determinants of antiviral or cytotoxic activities. However, the (1R)-diastereomer (10) was more resistant to glycosidic bond cleavage by thymidine phosphorylase than the (1S)-diastereomer (9).

Synthesis of the diastereomers of 5-(2,2-dichlorocyclopropyl)- and 5-(2-chlorocyclopropyl)-2'-deoxyuridine, and the antiviral and cytotoxic activity of these and bromo analogues.

Syntheses of the two diastereomers (5a and 6a) of 5-(2,2-dichlorocyclopropyl)-, and of the four diastereomers (7a-10a) of 5-(2-chlorocyclopropyl)-2'-deoxyuridine are described. These, and corresponding diastereomers (5b and 6b; 7b-10b) of 5-(2,2-dibromocyclopropyl)- and 5-(2-bromocyclopropyl)-2'-deoxyuridine (prepared in an earlier investigation) were examined for antiviral and cytotoxic activity, in comparison with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 5-fluoro-2'-deoxyuridine (FDU). 5-[(1R,2R)-2-Chlorocyclopropyl]-2'-deoxyuridine (9a) was the most active antiviral agent (IC50 = 25 micrograms/ml) against herpes simplex virus type 1 (HSV-1) relative to BVDU (IC50 = 0.082 microgram/ml). Compounds having the R configuration at the C-1 and/or C-2 positions of the 5-[2,2-dichloro(or 2-chloro)cyclopropyl] substituent exhibited the most potent antiviral activity. The cytotoxic activities of the 5-(2,2-dihalocyclopropyl)- (5-6a and b) and 5-(2-halocyclopropyl)- diastereomers (7-10a and b) were dependent upon both the configuration of the C-1 and/or C-2 cyclopropyl carbons and the nature of the halogeno (Cl, Br) substituent. 5-[(1R)-2,2-Dichlorocyclopropyl]-2'-deoxyuridine (6a) was the most active cytotoxic compound in the CCRF-CEM (IC50 = 17 microM) and HL-60 (IC50 = 64 microM) screens relative to FDU, which exhibited IC50 values of 4.7 x 10(-3) and 77 microM in these respective screens.

Parasite-induced processes for adenosine permeation in mouse erythrocytes infected with the malarial parasite Plasmodium yoelii.

In mouse erythrocytes harbouring the malarial parasite Plasmodium yoelii, three processes contributed to inward fluxes of adenosine, one of which is attributed to the native nucleoside transporter, because of the inhibitory effects of nitrobenzylthioinosine (NBMPR). New (parasite-induced) permeation processes of low NBMPR-sensitivity were (i) saturable fluxes with preference for the D enantiomer (D-Ado) and (ii) apparently unsaturable fluxes that proceeded by a channel-like route without enantiomeric selectivity. Parasite-induced fluxes of L- and D-Ado were similarly inhibited by furosemide [IC50 (concn. causing half-maximal inhibition) 15-17 microM], whereas D-Ado fluxes in uninfected erythrocytes were 10-fold less sensitive.

5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), a novel ligand with high affinity for polypeptides associated with nucleoside transport. Partial purification of the nitrobenzylthioinosine-binding protein of pig erythrocytes by affinity chromatography.

Derivatives of N6-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5'-linked derivatives of N6-(4-nitrobenzyl)adenosine (NBAdo) were evaluated as inhibitors of site-specific binding of [3H]nitrobenzylthioinosine (NBMPR) to pig erythrocyte membranes. Potent inhibitors were SAENTA [5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine] and acetyl-SAENTA (the 2-acetamidoethyl derivative of SAENTA). SAENTA was coupled to derivatized agarose-gel beads (Affi-Gel 10) to form an affinity matrix for chromatographic purification of NBMPR-binding polypeptides, which in pig erythrocytes are part of, or are associated with, the equilibrative nucleoside transporter. When pig erythrocyte membranes were solubilized with octyl glucoside (n-octyl beta-D-glucopyranoside) and applied to SAENTA-Affi-Gel 10 (SAENTA-AG10), polypeptides that migrated as a broad band on SDS/PAGE with an apparent molecular mass of 58-60 kDa were selectively retained by the affinity gel. These polypeptides were identified as components of the nucleoside transporter of pig erythrocytes by reactivity with a monoclonal antibody (mAb 11C4) that recognizes the NBMPR-binding protein of pig erythrocytes. Retention of the immunoreactive polypeptides by SAENTA-AG10 was blocked by NBAdo. The immunoreactive polypeptides were released from SAENTA-AG10 by elution under denaturing conditions with 1% SDS or by elution with detergent solutions containing competitive ligands (NBAdo or NBMPR). A 72-fold enrichment of the immunoreactive polypeptides was achieved by a single passage of solubilized, protein-depleted membranes through a column of SAENTA-AG10, followed by elution with detergent solutions containing NBAdo. These results demonstrate that polypeptide components of NBMPR-sensitive nucleoside-transport systems may be partly purified by affinity chromatography using gel media bearing SAENTA groups.

Drug-induced perturbations in the in vivo distribution of oncological radiotracers--II. 5-[125I]iodo-2'-deoxyuridine influenced by nitrobenzylthioinosine-5'-phosphate (NBMPR-P) and acyclothymidine (ACT).

Nitrobenzylthioinosine (NBMPR), a potent inhibitor of facilitated nucleoside transport in vitro and in vivo, and acyclothymidine (ACT), a potent inhibitor of pyrimidine nucleoside phosphorylase in vitro, have been used in an attempt to modulate the biodistribution of 125I-labelled iododeoxyuridine ([125I]IUdR). ACT or NBMPR-P (a water-soluble prodrug of NBMPR) were injected into BDF1 mice bearing implanted Lewis lung tumors, according to protocols which would provide high and low plasma levels of the inhibitor. Compared with controls, both inhibitors induced transient, marginal increases in hepatic, renal and blood levels of [125I]IUdR, and decreased levels in tumors at short time intervals after injection. It is concluded that there is a mild tumor-sparing effect when either NBMPR or ACT are administered together with single i.v. diagnostic doses of [125I]IUdR.

Enantiomeric selectivity of adenosine transport systems in mouse erythrocytes and L1210 cells.

In mediating the entry of adenosine into mouse erythrocytes and mouse leukaemia L1210 cells, nucleoside transport systems were stereoselective, showing a marked preference for the D-enantiomer of adenosine (D-Ado). Inward zero-trans fluxes of the mirror-image isomer, L-adenosine (L-Ado), in those cells were slow relative to those of D-Ado. Contributing to L-Ado fluxes in both cell types were (i) a transporter-mediated process of high nitrobenzylthioinosine-sensitivity and (ii) simple diffusion.

Interaction of [3H]dilazep at nucleoside transporter-associated binding sites on S49 mouse lymphoma cells.

Dilazep, a tertiary amine that is greater than 96% protonated at pH 7.4, is a potent inhibitor of facilitated diffusion (equilibrative) nucleoside transport (NT) in animal cells. In this study, saturable reversible binding of [3H]dilazep was demonstrated at sites on S49 mouse lymphoma cells but not in AE1 cells, an NT-deficient mutant of S49 cells. Mass law analysis of dilazep binding under equilibrium conditions revealed two saturable components, representing binding sites that differed about 50-fold in affinity for dilazep (Kd values of 0.21 and 10 nM). At pH 7.4, the low affinity sites were more abundant (Bmax, 3.5 X 10(5) sites/cell) than the high affinity site (Bmax, 3.0 X 10(4) sites/cell). Binding of dilazep was pH dependent; at pH 9.0, binding at the high affinity sites predominated, whereas, at pH 5.0, the low affinity component predominated, suggesting that these components represented binding of nonprotonated and protonated dilazep molecules, respectively. Nitrobenzylthioinosine (NBMPR) and physostigmine selectively blocked binding of nonprotonated and protonated species of dilazep, respectively, at pH 7.4, yielding Scatchard plots that were similar to control plots obtained at pH 5.0 and 9.0. First-order plots of the dissociation of [3H]dilazep-binding site complexes in the presence of excess nonradioactive dilazep at pH 7.4 were nonlinear and were resolved into rapid (rate constant, 3.4-4.7 min-1) and slow (rate constant, 0.13-0.15 min-1) components. In the presence of site-saturating concentrations of NBMPR or high concentrations of nucleoside permeants, dissociation of site-bound [3H]dilazep was incomplete and only the slow component of dissociation was apparent (rate constant, 0.11-0.19 min-1). The combined presence of nonradioactive dilazep and NBMPR yielded time courses of [3H]dilazep-site dissociation equivalent to those obtained in the presence of nonradioactive dilazep alone. These results are consistent with a model in which protonated and nonprotonated species of dilazep bind at separate sites on S49 cells. The absence of both high and low affinity sites on AE1 cells suggests that, in S49 cells, both populations of sites are associated with NT polypeptides. The high affinity sites that bind nonprotonated species of dilazep appear to overlap with NBMPR binding sites on these cells.

Nucleoside permeation in mouse erythrocytes infected with Plasmodium yoelii.

In normal mouse erythrocytes, nucleoside permeation was almost completely blocked in the presence of binding site-saturating concentrations of nitrobenzylthioinosine, whereas permeation in erythrocytes infected with the malarial parasite, Plasmodium yoelii, was substantial under these conditions, suggesting the presence of a permeation mechanism of low sensitivity to nitrobenzylthioinosine in the infected cells. Binding sites for nitrobenzylthioinosine were more numerous on infected erythrocytes than on uninfected cells. When mice infected with P. yoelii were treated with combinations of tubercidin and nitrobenzylthioinosine 5'-monophosphate, progression of parasitemia was delayed and survival times were increased.

[125I]iodohydroxynitrobenzylthioinosine: a new high-affinity nucleoside transporter probe.

[125I]iodohydroxynitrobenzylthioinosine ([125I]IH-NBMPR), a new gamma-labeled nucleoside transport inhibitor, has been prepared at a theoretical specific activity of 2000 Ci/mmol (1 Ci = 37 GBq). IH-NBMPR was more acidic than hydroxynitrobenzylthioinosine (H-NBMPR), having a pKa of 4.6. Site-specific binding of [125I]IH-NBMPR to membrane-enriched fractions (MEF) from S49 mouse lymphoma cells was pH dependent, increasing with the fraction of undissociated molecules present; it was maximal at pH 4.5 and negligible at pH 7.0. Scatchard analysis of specific binding to MEF from S49 cells under equilibrium conditions at pH 5.0 yielded a Kd of 15 nM (equivalent to 4.0 nM for the undissociated fraction of inhibitor molecules) and maximum number of binding sites (Bmax) of 4.9 pmol/mg protein. Specific binding of IH-NBMPR could not be demonstrated in MEF from AE1 cells, a nucleoside transport-deficient mutant of S49 cells. Influx of uridine into mouse erythrocytes at pH 5.0 in the presence of 5 microM IH-NBMPR (1.4 microM undissociated IH-NBMPR) was reduced to about 7% of the control value, indicating that this compound is an effective nucleoside transport inhibitor. Photoactivation of site-bound [125I]IH-NBMPR, following equilibration of the ligand with MEF from S49 cells at pH 5.0, resulted in specific covalent labeling of a polypeptide with a relative molecular mass of 52,000-63,000, identified on sodium dodecyl sulfate-polyacrylamide gels. These results indicate that the new, iodinated ligand is an inhibitor of nucleoside transport and that it binds specifically and with high affinity to nucleoside transporter polypeptides in mammalian cells.

Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells.

Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.