Testicular disposition of clofarabine in rats is dependent on equilibrative nucleoside transporters.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood-testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6-nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6-nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first-line treatment to avoid testicular relapse should be considered.

Multiple Computational Approaches for Predicting Drug Interactions with Human Equilibrative Nucleoside Transporter 1.

Equilibrativenucleoside transporters (ENTs) participate in the pharmacokinetics and disposition of nucleoside analog drugs. Understanding drug interactions with the ENTs may inform and facilitate the development of new drugs, including chemotherapeutics and antivirals that require access to sanctuary sites such as the male genital tract. This study created three-dimensional pharmacophores for ENT1 and ENT2 substrates and inhibitors using Kt and IC50 data curated from the literature. Substrate pharmacophores for ENT1 and ENT2 are distinct, with partial overlap of hydrogen bond donors, whereas the inhibitor pharmacophores predominantly feature hydrogen bond acceptors. Mizoribine and ribavirin mapped to the ENT1 substrate pharmacophore and proved to be substrates of the ENTs. The presence of the ENT-specific inhibitor 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (NBMPR) decreased mizoribine accumulation in ENT1 and ENT2 cells (ENT1, ∼70% decrease, P = 0.0046; ENT2, ∼50% decrease, P = 0.0012). NBMPR also decreased ribavirin accumulation in ENT1 and ENT2 cells (ENT1: ∼50% decrease, P = 0.0498; ENT2: ∼30% decrease, P = 0.0125). Darunavir mapped to the ENT1 inhibitor pharmacophore and NBMPR did not significantly influence darunavir accumulation in either ENT1 or ENT2 cells (ENT1: P = 0.28; ENT2: P = 0.53), indicating that darunavir's interaction with the ENTs is limited to inhibition. These computational and in vitro models can inform compound selection in the drug discovery and development process, thereby reducing time and expense of identification and optimization of ENT-interacting compounds. SIGNIFICANCE STATEMENT: This study developed computational models of human equilibrative nucleoside transporters (ENTs) to predict drug interactions and validated these models with two compounds in vitro. Identification and prediction of ENT1 and ENT2 substrates allows for the determination of drugs that can penetrate tissues expressing these transporters.

Behavioral effects of elevated expression of human equilibrative nucleoside transporter 1 in mice.

Adenosine concentrations are regulated by purinergic enzymes and nucleoside transporters. Transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1) have been generated (Parkinson et al., 2009 [7]). The present study tested the hypothesis that mice homozygous and heterozygous for the transgene exhibit differences in hENT1 mRNA and protein expression, and in behavioral responses to caffeine and ethanol, two drugs with adenosine-dependent actions. Real time polymerase chain reaction (PCR) was used to identify mice heterozygous and homozygous for the transgene. Gene expression, determined by real time PCR of cDNA reverse transcribed from cerebral cortex RNA, was 3.8-fold greater in homozygous mice. Protein abundance, determined by radioligand binding assays using 0.14nM [(3)H]S-(4-nitrobenzyl)-6-thioinosine ([(3)H]NBTI), was up to 84% greater in cortex synaptosome membranes from homozygous than from heterozygous mice. In western blots with an antibody specific for hENT1, a protein of approximately 40kDa was strongly labelled in cortex samples from homozygous mice, weakly labelled in samples from heterozygous mice and absent from samples from wild type mice. In behavioral assays, transgenic mice showed a greater response to ethanol and a reduced response to caffeine than wild type littermates; however, no significant differences between heterozygous and homozygous mice were detected. These data indicate that the difference in ENT1 function between wild type and heterozygous mice was greater than that between heterozygous and homozygous mice. Therefore, either heterozygous or homozygous hENT1 transgenic mice can be used in studies of ENT1 regulation of adenosine levels and adenosine dependent behaviors.

Expression of human equilibrative nucleoside transporter 1 in mouse neurons regulates adenosine levels in physiological and hypoxic-ischemic conditions.

Activation of adenosine A(1) receptors inhibits excitatory synaptic transmission. Equilibrative nucleoside transporters (ENTs) regulate extracellular adenosine levels; however, the role of neuronal ENTs in adenosine influx and efflux during cerebral ischemia has not been determined. We used mice with neuronal expression of human ENT type 1 and wild type (Wt) littermates to compare responses to in vitro hypoxic or ischemic conditions. Extracellular recordings in the CA1 region of hippocampal slices from transgenic (Tg) mice revealed increased basal synaptic transmission, relative to Wt slices, and an absence of 8-cyclopentyl-1,3-dipropyl-xanthine mediated augmentation of excitatory neurotransmission. Adenosine (10-100 µM) had a reduced potency for inhibiting synaptic transmission in slices from Tg mice; inhibitory concentration 50% values were approximately 25 and 50 µM in Wt and Tg slices, respectively. Potency of the A(1) receptor agonist N(6) -cyclopentyladenosine (1 nM-1 µM) was unchanged. Transient hypoxia or oxygen-glucose deprivation produced greater inhibition of excitatory neurotransmission in slices from Wt than Tg, mice. The ENT1 inhibitor S-(4-nitrobenzyl)-6-thioinosine abolished these differences. Taken together, our data provide evidence that neuronal ENTs reduce hypoxia- and ischemia-induced increases in extracellular adenosine levels and suggest that inhibition of neuronal adenosine transporters may be a target for the treatment of cerebral ischemia.

Determination of 6-benzylthioinosine in mouse and human plasma by liquid chromatography-tandem mass spectrometry.

This paper described the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 6-benzylthioinosine (6BT), a novel myeloid leukemia differentiation-inducing agent, in mouse and human plasma. In this method, 2-amino-6-benzylthioinosine (2A6BT) was used as internal standard and ethyl acetate was used as organic solvent for the extraction of 6BT and internal standard from plasma samples. The extracted samples were separated on YMC ODS-AQ((R)) column (2.0mmx50mm), and the eluates from the column were monitored by the positive-electrospray-ionization tandem mass spectrometer (ESI(+)-MS/MS). Quantification of 6BT by internal calibration with 2A6BT was carried out using multiple-reaction-monitoring (MRM) mode. This method had a linear calibration range of 3.00-1.00x10(3)ng/mL with correlation coefficients of 1.00 and 0.999 in mouse and human plasma. The lower limit of quantification (LLOQ) was 3.00ng/mL in either mouse or human plasma. The method had recovery of 6BT of 82-87% from mouse plasma and 90-98% from human plasma. Both accuracy (as percent error) and precision (as coefficient of variation) of the method were

Genetic interdependence of adenosine and dopamine receptors: evidence from receptor knockout mice.

Dopamine and adenosine receptors are known to share a considerable overlap in their regional distribution, being especially rich in the basal ganglia. Dopamine and adenosine receptors have been demonstrated to exhibit a parallel distribution on certain neuronal populations, and even when not directly co-localized, relationships (both antagonistic and synergistic) have been described. This study was designed to investigate dopaminergic and purinergic systems in mice with ablations of individual dopamine or adenosine receptors. In situ hybridization histochemistry and autoradiography was used to examine the level of mRNA and protein expression of specific receptors and transporters in dopaminergic pathways. Expression of the mRNA encoding the dopamine D2 receptor was elevated in the caudate putamen of D1, D3 and A2A receptor knockout mice; this was mirrored by an increase in D2 receptor protein in D1 and D3 receptor knockout mice, but not in A2A knockout mice. Dopamine D1 receptor binding was decreased in the caudate putamen, nucleus accumbens, olfactory tubercle and ventral pallidum of D2 receptor knockout mice. In substantia nigra pars compacta, dopamine transporter mRNA expression was dramatically decreased in D3 receptor knockout mice, but elevated in A2A receptor knockout mice. All dopamine receptor knockout mice examined exhibited increased A2A receptor binding in the caudate putamen, nucleus accumbens and olfactory tubercle. These data are consistent with the existence of functional interactions between dopaminergic and purinergic systems in these reward and motor-related brain regions.

Interaction of the novel adenosine uptake inhibitor 3-[1-(6,7-diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345) with the es and ei subtypes of equilibrative nucleoside transporters.

Nucleosides such as adenosine, as well as many nucleoside-based drugs, permeate cell membranes via a family of equilibrative nucleoside transporters (ENTs). We assessed the effects of (3-[1-(6,7-diethoxy-2-morpholino-quinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345), a novel anti-inflammatory agent that potentiates the actions of adenosine, on the es (inhibitor-sensitive) and ei (inhibitor-resistant) subtypes of ENTs in human, mouse, and rat cells. KF24345 was similar to the prototypical high-affinity inhibitor nitrobenzylthioinosine (NBMPR) for blocking the human es transporter (K(I) of approximately 0.4 nM), but was 50-fold more effective than NBMPR at blocking the human ei transporter (K(I) of approximately 100 nM). KF24345 displayed significantly less species heterogeneity in its affinity for the es transporter than did dipyridamole, a widely used inhibitor of nucleoside transport; KF24345 may thus prove useful as an inhibitor for studies of nucleoside metabolism in a range of animal models. Furthermore, KF24345 seemed to act as a noncompetitive inhibitor of both [(3)H]NBMPR binding and [(3)H]nucleoside uptake by human es transporters, and these kinetics were consistent with an observed slow dissociation of KF24345 from the inhibitor binding site. KF24345 also exhibited unusual biphasic profiles for inhibition of [(3)H]NBMPR binding to membranes prepared from a recombinant human es transporter model (PK15-hENT1), suggesting the presence of multiple populations of NBMPR binding proteins in these membranes. The atypical tight binding interaction of KF24345 with the es transporter may prove useful for the molecular delineation of inhibitor binding domains and will facilitate its use as an in vivo inhibitor of nucleoside transport in studies focused on the biological effects of adenosine.

Uptake of nitrobenzylthioinosine and purine beta-L-nucleosides by intracellular Toxoplasma gondii.

Intracellular Toxoplasma gondii grown in human foreskin fibroblast cells transported nitrobenzylthioinosine [NBMPR; 6-[(4-nitrobenzyl)mercapto]-9-beta-D-ribofuranosylpurine], an inhibitor of nucleoside transport in mammalian cells, as well as the nonphysiological beta-L-enantiomers of purine nucleosides, beta-L-adenosine, beta-L-deoxyadenosine, and beta-L-guanosine. The beta-L-pyrimidine nucleosides, beta-L-uridine, beta-L-cytidine, and beta-L-thymidine, were not transported. The uptake of NBMPR and the nonphysiological purine nucleoside beta-L-enantiomers by the intracellular parasites also implies that Toxoplasma-infected cells can transport these nucleosides. In sharp contrast, under the same conditions, uninfected fibroblast cells did not transport NBMPR or any of the unnatural beta-L-nucleosides. beta-D-Adenosine and dipyridamole, another inhibitor of nucleoside transport, inhibited the uptake of NBMPR and beta-L-stereoisomers of the purine nucleosides by intracellular Toxoplasma and Toxoplasma-infected cells. Furthermore, infection with a Toxoplasma mutant deficient in parasite adenosine/purine nucleoside transport reduced or abolished the uptake of beta-D-adenosine, NBMPR, and purine beta-L-nucleosides. Hence, the presence of the Toxoplasma adenosine/purine nucleoside transporters is apparently essential for the uptake of NBMPR and purine beta-L-nucleosides by intracellular Toxoplasma and Toxoplasma-infected cells. These results also demonstrate that, in contrast to the mammalian nucleoside transporters, the Toxoplasma adenosine/purine nucleoside transporter(s) lacks stereospecificity and substrate specificity in the transport of purine nucleosides. In addition, infection with T. gondii confers the properties of the parasite's purine nucleoside transport on the parasitized host cells and enables the infected cells to transport purine nucleosides that were not transported by uninfected cells. These unique characteristics of purine nucleoside transport in T. gondii may aid in the identification of new promising antitoxoplasmic drugs.

Comparative pharmacokinetics of oral 6-mercaptopurine and intravenous 6-mercaptopurine riboside in children.

BACKGROUND: The poor absorption of orally administered 6-mercaptopurine (6MP) causes a wide variation in its cytotoxic efficacy. An i.v. dosage form would eliminate this problem. Our objective was to compare the pharmacokinetics of 6MP administered orally with those of an i.v. dosage form 6-mercaptopurine riboside (6MPR), in children with acute lymphoblastic leukemia or malignant lymphoma. METHODS: A total of 10 children were treated with oral 6MP, 50 mg/m(2) per day, while five children were treated with 6MPR, 50 mg/m(2) per day, administered by rapid i.v. injection. The plasma concentrations of 6MP and of 6MPR were measured on day 0, while the concentrations of 6-thioguanine nucleotides (6TGN) in red blood cells (RBC) were measured on day 2. The area under the plasma concentration-time curve (AUC1-5) was calculated from 1 to 5 h after drug administration. RESULTS: With the intravenously administered 6MPR, the AUC1-5 ranged from 124 to 186 (1.5-fold range, median 145) microM min; only two samples were obtained for the RBC concentration of 6TGN, and were 121 and 273 pmol per 25 mg hemoglobin. With the orally administered 6MP, the AUC1-5 ranged from 23 to 65 microM min (2.8-fold range, median 56); the RBC concentration of 6TGN ranged from 18 to 152 pmol per 25 mg hemoglobin (median 75). CONCLUSION: The i.v. administration of 6MPR showed less interindividual variation in the AUC1-5 coupled with a higher RBC level of 6TGN as compared with those by oral 6MP. We conclude that the i.v. administration of 6MPR achieves stable blood levels of active drug in children undergoing cancer chemotherapy.

Ontogenic and longitudinal activity of Na(+)-nucleoside transporters in the human intestine.

The objectives of our study were to identify the types of nucleoside transporters present in the human fetal small intestine and to characterize their developmental activity, longitudinal distribution, and transport kinetics compared with those present in the adult intestine. Nucleoside uptake by intestinal brush-border membrane vesicles was measured by an inhibitor-stop rapid filtration technique. Only the purine-specific (N1; hCNT2) and the pyrimidine-specific (N2; hCNT1) Na(+)-dependent nucleoside transporters were found to be present on the brush-border membranes of the enterocytes along the entire length of the fetal and adult small intestines. The activity of these transporters was higher in the proximal than in the distal small intestine. Both the N1 and N2 transporters found in the fetal intestine shared similar kinetic properties (Michaelis-Menten constant and Na(+)-nucleoside stoichiometry) to those in the adult intestine. During the period of rapid morphogenesis (11-15 wk gestation), no temporal differences were apparent in the activity of the N1 and N2 transporters in the fetal small intestine. These findings have implications for the absorption of drugs from the amniotic fluid by the fetus after maternal drug administration of nucleoside drugs such as the antivirals zidovudine and didanosine.

Time development and regional distribution of [3H]nitrobenzylthioinosine adenosine uptake site binding in the mouse brain after acute Pentylenetetrazol-induced seizures.

Adenosine has been shown to play a significant role as a modulator of neuronal activity in convulsion disorders, acting as an endogenous anticonvulsant agent. In the present study, we have investigated in mice the effect of acute tonic-clonic seizures induced by a single Pentylenetetrazol (PTZ)-injection (a) on the time development of adenosine uptake site binding after seizures in membranes of hippocampus, cortex, cerebellum, and striatum, and (b) on the regional distribution of adenosine uptake sites in the mouse brain by using "in vitro" quantitative autoradiography. As radioligand, the specific adenosine uptake blocker [3H]N-9-nitrobenzylthioinosine ([3H]NBI) was used. Acute seizures induced a rapid significant increase in [3H]NBI uptake site binding in hippocampus and cerebellum within 5 min, in cortex within 10 min after seizures, which reached a maximum level at 1 hr and reversed to control levels in about 150 min after seizures. On the contrary, in striatum a significant decrease of [3H]NBI uptake site binding was observed within 10 min after seizures, which reached its maximum at 1 hr and reversed to control levels at 150 min after seizures. With this single exception of striatum the "in vitro" quantitative autoradiography revealed a rather widespread upregulation of [3H]NBI uptake site density in the mouse brain, which was specifically enhanced in certain areas known to mediate seizure activity, such as hippocampus, specific thalamic nuclei, temporal cortex, and substantia nigra. The pattern of increases in [3H]NBI uptake site binding as they develop after acute seizures correlates well in time with the rapid enhancement of endogenous adenosine concentration released during epileptic activity. Since extracellular adenosine levels seem to be regulated by a rapid reuptake system, it seems likely that in our study, the [3H]NBI adenosine uptake system is acutely activated by seizures in order to compensate for the excess of endogenous adenosine. Furthermore, the upregulation of [3H]NBI uptake sites as revealed by the "in vitro" quantitative autoradiography seems to be organized in selective brain areas related to seizure propagation.

Lower density of A1 adenosine receptors in nucleus reticularis thalami in rats with genetic absence epilepsy.

The possible involvement of the adenosinergic modulatory system in the pathogenesis of absence seizures was investigated in genetic absence epilepsy rats from Strasbourg (GAERS). Using in vitro quantitative autoradiography, the distribution of A1 adenosine receptors and adenosine uptake sites in the brain of GAERS was studied and compared to that of control animals. An area-specific lower density of A1 receptors (15% decrease) was detected in reticular (nRT) and anterior ventral (AV) thalamic nuclei as well as basal ganglia in the brains of GAERS animals compared with control animals. Since adenosine exerts an anti-oscillatory effect on the thalamic nuclei by suppressing (via A1 receptors) excitatory as well as inhibitory neurotransmitter release, the impairment in A1 receptor density seen here, especially in nRT, could be implicated in the thalamic rhythmicity underlying spike and wave discharges present in this absence epilepsy model.

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.

Ability of nitrobenzylthioinosine to cross the blood-brain barrier in rats.

Nucleoside transport inhibitors that cross the blood-brain barrier may be able to potentiate the neuroprotective effects of adenosine. We tested whether nitrobenzylthioinosine (NBMPR) crosses the blood-brain barrier in three types of experiments. First, intravenous injection of [3H]NBMPR and [14C]sucrose was performed. Brain volume of distribution and brain delivery were greater for [3H]NBMPR than for [14C]sucrose. Second, rats were injected intraperitoneally with NBMPR 5'-monophosphate (NBMPR-P), a prodrug form of NBMPR, or vehicle. Perchloric acid extracts of brains from rats treated with NBMPR-P inhibited [3H]NBMPR binding in competition binding assays nearly 3-fold more than extracts from brains of vehicle-treated animals. Third, cerebrospinal fluid (CSF) extracted from rats treated with NBMPR-P (10 mg/kg i.p.) contained 24.1 +/- 4.4 nM NBMPR while levels were undetectable in CSF from vehicle-treated rats. From these data, we conclude that NBMPR crosses the blood-brain barrier.

Phase I trial of fluorouracil modulation by N-phosphonacetyl-L-aspartate and 6-methylmercaptopurine ribonucleoside.

Inhibition of pyrimidine and purine synthesis has been demonstrated to potentiate 5-fluorouracil (5-FU) activity in preclinical models. Low-dose phosphonacetyl-L-aspartate (PALA) potentiates the incorporation of 5-FU into RNA, without detectably increasing its toxicity. 6-Methylmercaptopurine riboside (MMPR) results in inhibition of purine biosynthesis with elevation of phosphoribosyl pyrophosphate (PRPP), which in turn is believed to increase the phosphorylation and intracellular retention of 5-FU. We conducted a phase I clinical trial to determine the maximum tolerated dose of 5-FU in combination with low-dose PALA and a biochemically-optimized dose of MMPR. The regimen consisted of PALA 250 mg/m2 given on day 1, followed 24 h later by MMPR 150 mg/m2, and escalating doses of 5-FU from 1625 to 2600 mg/m2 by 24 h continuous infusion. This regimen was repeated weekly. A group of 29 patients with a diagnosis of malignant solid tumor were entered; their median performance status was 1. The dose-limiting toxicity was mucositis, while other gastrointestinal toxicity was minimal. Two patients also experienced ischemic chest pain during the 5-FU infusion. The maximum tolerated dose of 5-FU in this combination was 2600 mg/m2. Several responses were observed including a complete remission in a previously treated breast cancer patient and two partial responses in breast and colon cancer. MMPR pharmacokinetics were obtained from urine analyses in 21 patients on this trial; there was no correlation between the pharmacokinetics of MMPR and the toxicity observed. This regimen was well tolerated and phase II trials are warranted using PALA 250 mg/m2, MMPR 150 mg/m2, and 5-FU 2300 mg/m2 by continuous infusion over 24 h.

DNA modification induced by 6-mercaptopurine riboside in murine embryos.

The cytostatic drug 6-mercaptopurine riboside (6-MPr) was investigated in mice in order to test the hypothesis that the teratogenicity of this antimetabolite is paralleled by an incorporation into the DNA of the embryos during organogenesis. DNA modification in the embryos was analysed 4 h following s.c. administration of [35S]-labelled 6-MPr to the dams on day 11 of pregnancy. The DNA of the embryos was isolated and hydrolysed to the bases by formic acid. Following separation by cation-exchange HPLC 6-thioguanine was found in the hydrolysate. Quantitation was performed by liquid scintillation counting. Evaluations of 6 doses in the range of 8-25 mg/kg were performed. An incorporation rate of 6-thioguanine from 32-56 pmol per mumol guanine was found in the DNA of the embryos. These findings suggest that, similar to the previously studied alkylating agents, the teratogenicity of 6-MPr may be, at least in part, induced via DNA modification of the embryos.

Adenosine 5'-triphosphate modulation of nitrobenzylthioinosine binding sites in plasma membranes of bovine chromaffin cells.

Nitrobenzylthioinosine (NBTI) is a high affinity probe for facilitated diffusion nucleoside transporters. Kinetic analysis of the binding of [3H]NBTI to plasma membranes of chromaffin cells was conducted in the presence or absence of adenosine 5'-triphosphate (ATP). Similar curvilinear plots with a Hill number of 1.32 were obtained in both conditions. ATP significantly increased the number of NBTI binding sites in these preparations showing Bmax values of 1.62 +/- 0.20 pmol/mg protein for controls and 3.22 +/- 0.31 pmol/mg protein in the presence of ATP. However, the affinity constant (KD) was not significantly modified. The non-metabolizable ATP analogue, 5'-adenylyl imidodiphosphate (AMP-PNP) and diadenosine tetraphosphate (Ap4A) can mimic the stimulatory ATP effect, but adenosine monophosphate (AMP) has no effect on the NBTI binding to plasma membranes. These results indicate a modulatory role for ATP, non-hydrolysis dependent, on nucleoside transport in chromaffin cells. Therefore, a nucleotide binding site on the nucleoside transporter similar to that described for glucose transporter could be suggested.

In vivo effectiveness of several nucleoside transport inhibitors in mice and hamsters.

The in vivo nucleoside transport inhibitory effects of 6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside (NBMPR), used as its 5'-monophosphate derivative (NBMPR-P), dilazep, mioflazine and its derivatives soluflazine, R57974 and R75231, were investigated in BALB/c mice. The extent and duration of action were followed by assaying adenosine transport in blood cells sampled at time intervals following i.p. administration (ca. 20 mg/kg). Dilazep and R57974 were found to be short-acting inhibitors, while NBMPR-P and R75231 were similar in their action and caused essentially full inhibition of adenosine transport over a 4- to 5-h period. Mioflazine and soluflazine were rather ineffective, causing only partial inhibition. R75231 was also active after oral administration which, when repeated three times in 4-h intervals, resulted in essentially full transport inhibition up to 20 h following the initial dose. Effects of NBMPR-P, R57974 and dilazep on adenosine transport in blood cells were also measured in blood cells of hamsters after i.p. administration of the same doses. All three drugs caused full transport inhibition, but the action of dilazep and R75231 showed reversal within about 30 min and 2 h, respectively, while NBMPR-P caused full inhibition for at least 3-4 h. These results demonstrate the potential of the mioflazine derivative R75231 to be useful in vivo, possibly even after p.o. administration, for host protection against the actions of cytotoxic nucleosides used in experimental antiparasitic therapy or other studies requiring suppression of nucleoside transport.

Solubilization and reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.

Uptake of [3H]uridine by Ehrlich cells was mediated by both nitrobenzylthioinosine (NBMPR)-sensitive (75%) and NBMPR-insensitive (25%) mechanisms. Each cell contained approx. 26,000 high-affinity (KD = 0.19 nM) recognition sites for [3H]NBMPR, and binding was inhibited by dipyridamole and adenosine at concentrations similar to those required for inhibition of [3H]uridine uptake. Calculations show that each cell contains a total of about 35,000 nucleoside transporters. Photoaffinity labelling of a partially purified preparation of plasma membranes with [3H]NBMPR resulted in a single broad 3H-labelled band on SDS/polyacrylamide gels, with an apparent molecular-mass peak of 42 kDa. This is in contrast with human erythrocyte membranes, where [3H]NBMPR photolabelled two broad bands with peaks at 55 and 80 kDa. Treatment of photoaffinity-labelled membranes with endoglycosidase F decreased the apparent molecular masses of both the Ehrlich-cell and erythrocyte [3H]NBMPR-labelled proteins to approx. 40 kDa. These results suggest that the human erythrocyte [3H]NBMPR-binding polypeptides are more extensively glycosylated than the corresponding Ehrlich-cell polypeptides. Octyl beta-D-glucopyranoside [1.0% (w/v) + asolectin] solubilized over 90% of the [3H]NBMPR-binding sites, with near-complete retention of [3H]NBMPR-binding characteristics. The only major change was a 65-fold decrease in affinity for dipyridamole, which was partly reversed upon incorporation of the solubilized proteins into asolectin membranes. Proteoliposomes, prepared by using asolectin and the octyl glucoside-solubilized plasma membranes, were capable of accumulating [3H]uridine via a protein-dependent dipyridamole/nitrobenzylthioguanosine/dilazep-sensitive mechanism. We have thus demonstrated the efficient solubilization and functional reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.

[Effect of nitrobenzylthioinosine accumulated by the heart in rats and guinea pigs on the release of adenine nucleotide breakdown products during ischemia and reperfusion]. / Vliianie nitrobenziltioinozina, nakoplennogo serdtsem krys i morskikh svinok, na vysvobozhdenie produktov raspada adeninnukleotidov pri ishemii i reperfuzii.

The uptake kinetics of nitrobenzyl thioinosine (NBTI), a nucleoside transport inhibitor, was studied in the isolated Langendorf-perfused guinea pig and rat hearts. In rats the rate constant of NBTI uptake was higher and the extent of NBTI accumulation was less than in guinea pig hearts. Heart-accumulated NBTI inhibited the total release of adenine nucleotide degradation products (ANDP) during reperfusion 25 min after global ischemia. The effect was more pronounced in guinea-pig hearts-in accordance with observed higher myocardial concentration of NBTI. Unlike other ANDP, the release adenosine by guinea-pig hearts was unchanged and that by rat hearts increased. In spite of significant NBTI-induced decrease of ANDP losses recovery of contractile function during reperfusion was not observed to improve.