Hypoxanthine uptake by skeletal muscle microvascular endothelial cells from equilibrative nucleoside transporter 1 (ENT1)-null mice: effect of oxidative stress.

Adenosine is an endogenous regulator of vascular tone. This activity of adenosine is terminated by its uptake and metabolism by microvascular endothelial cells (MVEC). The predominant transporter involved is ENT1 (equilibrative nucleoside transporter subtype 1). MVEC also express the nucleobase transporter (ENBT1) which is involved in the cellular flux of adenosine metabolites such as hypoxanthine. Changes in either of these transport systems would impact the bioactivity of adenosine and its metabolism, including the formation of oxygen free radicals. MVEC isolated from skeletal muscle of ENT1(+/+) and ENT1(-/-) mice were subjected to oxidative stress induced by simulated ischemia/reperfusion or menadione. The functional activities of ENT1 and ENBT1 were assessed based on zero-trans influx kinetics of radiolabeled substrates. There was a reduction in the rate of ENBT1-mediated hypoxanthine uptake by ENT1(+/+) MVEC treated with menadione or after exposure to conditions that simulate ischemia/reperfusion. In both cases, the superoxide dismutase mimetic MnTMPyP attenuated the loss of ENBT1 activity, implicating superoxide radicals in the response. In contrast, MVEC isolated from ENT1(-/-) mice showed no reduction in ENBT1 activity upon treatment with menadione or simulated ischemia/reperfusion, but they did have a significantly higher level of catalase activity relative to ENT1(+/+) MVEC. These data suggest that ENBT1 activity is decreased in MVEC in response to the increased superoxide radical that is associated with ischemia/reperfusion injury. MVEC isolated from ENT1(-/-) mice do not show this reduction in ENBT1, possibly due to increased catalase activity.

Calculations of molecular properties in hybrid coupled-cluster and molecular mechanics approach.

We report benchmark calculations obtained with our new coupled-cluster singles and doubles (CCSD) code for calculating the first- and second-order molecular properties. This code can be easily incorporated into combined [Valiev, M.; Kowalski, K. J. Chem. Phys. 2006, 125, 211101] classical molecular mechanics (MM) and ab initio coupled-cluster (CC) calculations using NWChem, enabling us to study molecular properties in a realistic environment. To test this methodology, we discuss the results of calculations of dipole moments and static polarizabilities for the Cl2O system in the CCl4 solution using the CCSD (CC with singles and doubles) linear response approach. We also discuss the application of the asymptotic extrapolation scheme (AES) [Kowalski, K.; Valiev, M. J. Phys. Chem. A 2006, 110, 13106] in reducing the numerical cost of CCSD calculations.

Increased L-arginine uptake and inducible nitric oxide synthase activity in aortas of rats with heart failure.

L-Arginine crosses the cell membrane primarily through the system y(+) transporter. The aim of this study was to investigate the role of L-arginine transport in nitric oxide (NO) production in aortas of rats with heart failure induced by myocardial infarction. Tumor necrosis factor-alpha levels in aortas of rats with heart failure were six times higher than in sham rats (P < 0.01). L-Arginine uptake was increased in aortas of rats with heart failure compared with sham rats (P < 0.01). Cationic amino acid transporter-2B and inducible (i) nitric oxide synthase (NOS) expression were increased in aortas of rats with heart failure compared with sham rats (P < 0.05). Aortic strips from rats with heart failure treated with L-arginine but not D-arginine increased NO production (P < 0.05). The effect of L-arginine on NO production was blocked by L-lysine, a basic amino acid that shares the same system y(+) transporter with L-arginine, and by the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Treatment with L-lysine and L-NAME in vivo decreased plasma nitrate and nitrite levels in rats with heart failure (P < 0.05). Our data demonstrate that NO production is dependent on iNOS activity and L-arginine uptake and suggest that L-arginine transport plays an important role in enhanced NO production in heart failure.

Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain.

Mammalian cells express at least two subtypes of equilibrative nucleoside transporters, i.e. ENT1 and ENT2, which can be distinguished functionally by their sensitivity and resistance respectively to inhibition by nitrobenzylthioinosine. The ENT1 transporters exhibit distinctive species differences in their sensitivities to inhibition by dipyridamole, dilazep and draflazine (human>mouse>rat). A comparison of the ENT1 structures in the three species would facilitate the identification of the regions involved in the actions of these cardioprotective agents. We now report the molecular cloning and functional expression of the murine (m)ENT1 and mENT2 transporters. mENT1 and mENT2 encode proteins containing 458 and 456 residues respectively, with a predicted 11-transmembrane-domain topology. mENT1 has 88% and 78% amino acid identity with rat ENT1 and human ENT1 respectively; mENT2 is more highly conserved, with 94% and 88% identity with rat ENT2 and human ENT2 respectively. We have also isolated two additional distinct cDNAs that encode proteins similar to mENT1; these probably represent distinct mENT1 isoforms or alternative splicing products. One cDNA encodes a protein with two additional amino acids (designated mENT1b) that adds a potential protein kinase CK2 phosphorylation site in the central intracellular loop of the transporter, and is similar, in this regard, to the human and rat ENT1 orthologues. The other cDNA has a 5'-untranslated region sequence that is distinct from that of full-length mENT1. Microinjection of mENT1, mENT1b or mENT2 cRNA into Xenopus oocytes resulted in enhanced uptake of [(3)H]uridine by the oocytes relative to that seen in water-injected controls. mENT1-mediated, but not mENT2-mediated, [(3)H]uridine uptake was inhibited by nitrobenzylthioinosine and dilazep. Dipyridamole inhibited both mENT1 and mENT2, but was significantly more effective against mENT1. Adenosine inhibited both systems with a similar potency, as did a range of other purine and pyrimidine nucleosides. These results are compatible with the known characteristics of the native mENT1 and mENT2 transporters.

Interaction of a series of draflazine analogues with equilibrative nucleoside transporters: species differences and transporter subtype selectivity.

The equilibrative nucleoside transporters of mammalian cells play an important role in the regulation of extracellular adenosine concentrations, and inhibition of these transporters potentiates the biological effects of adenosine. Two subtypes of equilibrative transporters have been defined by their differential sensitivities to inhibition by nitrobenzylthioinosine (NBMPR; es/ENT1, sensitive; ei/ENT2, insensitive). In addition, significant species differences have been noted in es/ENT1 transporter affinity for a subset of inhibitors including draflazine and dipyridamole. Draflazine and a series of 15 chemically related compounds were compared for their abilities to: (a) inhibit the binding of [3H]NBMPR to the es/ENT1 transporter in mouse Ehrlich cell and human erythrocyte membranes, and (b) inhibit the es/ENT1 and ei/ENT2 transporter-mediated uptake of [3H]uridine in Ehrlich cells. Compounds within this series represented over a 1000-fold range of affinities for the es/ENT1 and ei/ENT2 transporters with subtype selectivities (ENT1/ENT2) ranging from 370 for R70527 to 0.17 for soluflazine. Five other analogues were identified, in addition to soluflazine, that had significantly higher affinity for the ei/ENT2 transporter compared with es/ENT1. Structure activity analyses of these data identified the requirement of a hydrophobic group connected to a 2-aminocarbonyl piperazine by a 5-carbon chain for high-affinity interactions with es/ENT1. This hydrophobic moiety was not as important for ei/ENT2 affinity and, in contrast to es/ENT1, a shorter alkyl chain enhanced binding to ei/ENT2. These draflazine analogues also varied in their differential affinities for mouse vs. human es/ENT1 transporters, and the degree of species discrimination was strongly dependent on the position of the aminocarbonyl group on the piperazine ring. This information, combined with structural data derived from molecular studies with ENT1 and ENT2 recombinant proteins, should guide further development of subtype-selective inhibitors of the equilibrative nucleoside transporters.

[3H]gemcitabine uptake by nucleoside transporters in a human head and neck squamous carcinoma cell line.

Cellular uptake of many chemotherapeutic nucleoside analogs is dependent on the activity of a family of nucleoside transport proteins located in the cell plasma membrane. In the present study, we examined the role of these transporters in the accumulation of gemcitabine by a human head and neck squamous carcinoma cell line. The uptake of [3H]gemcitibine was compared with that of [3H]uridine and [3H]formycin B in the parent cell line (HN-5a) and in a gemcitabine-resistant variant (GEM-8e). The HN-5a and GEM-8e cells were similar in their transport characteristics and expressed predominantly the es (equilibrative, inhibitor-sensitive) transporter subtype; less than 10% of the influx of [3H]formycin B or [3H]uridine was mediated by the ei (equilibrative inhibitor-resistant) system, and there was no evidence for Na+-dependent nucleoside transporters. [3H]Gemcitabine (10 microM) entered these cells via both the es and ei transporters with an initial rate of uptake similar to that seen with the use of [3H]formycin B or [3H]uridine. In addition, ATP-replete cells accumulated significantly less [3H]gemcitabine than did ATP-depleted cells, which is indicative of an active efflux mechanism for gemcitabine. These results show that gemcitabine is a substrate for both the es and ei nucleoside transporters of HN-5a and GEM-8e cells and that gemcitabine resistance of the GEM-8e cells cannot be attributed to changes in transporter activity. Further studies to define the characteristics of the putative efflux mechanism are clearly warranted because this system has the potential to significantly affect the clinical efficacy of gemcitabine.

Demonstration of equilibrative nucleoside transporters (hENT1 and hENT2) in nuclear envelopes of cultured human choriocarcinoma (BeWo) cells by functional reconstitution in proteoliposomes.

The equilibrative nucleoside transporters (ENTs) are a newly recognized family of membrane proteins of which hENT1 is the nitrobenzylmercaptopurine ribonucleoside (NBMPR)-sensitive (es) and hENT2 the NBMPR-insensitive (ei) transporter of human cells. BeWo cells exhibit large numbers (>10(7)/cell) of NBMPR-binding sites and high es and ei nucleoside transport activities relative to other cell types. In this work, we have demonstrated that proliferating BeWo cells possess (i) mRNA encoding hENT1 and hENT2 and (ii) hENT1-specific immunoepitopes. We examined NBMPR binding and its inhibition of uridine transport in various BeWo membrane fractions and proteoliposomes derived therefrom to determine if NBMPR binding to intracellular membranes represented interaction with functional es transporters. Unfractionated membranes and fractions enriched 5-fold in plasma membranes relative to postnuclear supernatants exhibited high NBMPR binding activity. Intact nuclei and nuclear envelopes also exhibited abundant quantities of NBMPR-binding sites with affinities similar to those of enriched plasma membranes (Kd = 0.4-0.9 nM). When proteoliposomes were made from octyl glucoside-solubilized membranes, high affinity NBMPR-binding sites were not only observed in crude membrane preparations and plasma membrane-enriched fractions but also in nuclear envelope fractions. Proteoliposomes prepared from either unfractionated membranes or nuclear envelopes exhibited both hENT1-mediated (82-85%) and hENT2-mediated (15-18%) transport of [3H]uridine. These results provided evidence for the presence of functional es and ei transporters in nuclear membranes and endoplasmic reticulum, suggesting that hENT1 and hENT2 may function in the translocation of nucleosides between the cytosol and the luminal compartments of one or both of these membrane types.

Interaction of 2',2'-difluorodeoxycytidine (gemcitabine) and formycin B with the Na+-dependent and -independent nucleoside transporters of Ehrlich ascites tumor cells.

The uptake of [3H]formycin B by Ehrlich ascites tumor cells was examined in both normal Na+ buffer (physiological) and nominally Na+-free buffer (iso-osmotic replacement with Li+). These studies were conducted to further characterize the equilibrative nucleoside transporter subtypes of Ehrlich cells and to assess the contribution of Na+-dependent concentrative transport mechanisms to the cellular accumulation of nucleoside analogues by these cells. Formycin B is poorly metabolized by mammalian cells and, hence, can be used as a substrate to measure transport kinetics in energetically competent cells. Initial studies established that formycin B inhibited [3H]uridine uptake by the ei (equilibrative inhibitor-insensitive) and es (equilibrative inhibitor-sensitive) transporters of Ehrlich cells with Ki values of 48 +/- 28 and 277 +/- 25 microM, respectively. Similarly, [3H]formycin B had Km values of 111 +/- 52 and 635 +/- 147 microM for uptake by the ei and es transporters, respectively. When assays were conducted in the presence of Na+, plus 100 nM nitrobenzylthioinosine to prevent efflux via the es transporters, the intracellular concentration of [3H]formycin B exceeded the initial medium concentration by more than 3-fold, indicating the activity of a Na+-dependent transporter. Interestingly, the initial rate of uptake of [3H]formycin B was significantly higher in the Li+ buffer (es-mediated Vmax = 65 +/- 10 pmol/microliter . sec) than in the Na+ buffer (Vmax = 8.4 +/- 0.9 pmol/microliter . sec); this may reflect trans-acceleration of [3H]formycin B uptake by elevated intracellular adenosine levels resulting from the low Na+ environment. This model was then used to assess the interaction of gemcitabine (2',2'-difluorodeoxycytidine) with the equilibrative and concentrative nucleoside transporters. Gemcitabine, which has shown considerable potential for the treatment of solid tumors, was a relatively poor inhibitor of [3H]formycin B uptake via the equilibrative transporters (IC50 approximately 400 microM). In contrast, gemcitabine was a potent inhibitor of the Na+-dependent nucleoside transporter of Ehrlich cells (IC50 = 17 +/- 5 nM). These results suggest that the cellular expression/activity of Na+-dependent nucleoside transporters may be an important determinant in gemcitabine cytotoxicity and clinical efficacy.

The construction and application of a lux-based nitrate biosensor.

A plasmid-borne transcriptional fusion between the Escherichia coli nitrate reductase (narG) promoter and the Photorhabdus luminescens lux operon provides E. coli with a highly bioluminescent phenotype in the presence of nitrate. This E. coli biosensor can detect nitrate to a level of 5 x 10(-5) mol l-1 (0.3 ppm), levels relevant to those levels encountered in brewing water. Since induction of the narG promoter requires NarL, the plasmid-based sensor can also be used to interrogate enteric bacteria for the presence of functional homologues of this E. coli regulatory protein. Obesumbacterium proteus, an important bacterial brewery contaminant, failed to provide nitrate-dependent bioluminescence demonstrating divergence in this organism from E. coli in the mechanism of nitrate reductase regulation.

Enhancement of the functional stability of solubilized nucleoside transporters by substrates and inhibitors.

Purification of functional nucleoside transporters has been hampered by the instability of detergent-solubilized proteins. The present study was undertaken to determine if the presence of specific transporter ligands in the solubilization medium could enhance the functional stability of the isolated proteins. Ehrlich cell plasma membranes were solubilized with 1% (w/v) octylglucoside (+/- transporter ligands) and reconstituted into liposomal membranes either immediately after solubilization or after storage for 48 hr at 6 degrees. Storage resulted in a parallel loss (approximately 60%) of [3H]nitrobenzylthioinosine (NBMPR) binding and reconstituted [3H]uridine uptake activities. furthermore, upon storage, the relative amount of NBMPR-resistant [3H]uridine uptake by the reconstituted system dropped from 19 +/- 2 to 8 +/- 1% of the total mediated influx. The inclusion of high concentrations (> 10 mM) of adenosine in the solubilization medium completely prevented the storage-induced loss of both [3H]NBMPR binding and [3H]uridine influx activity, and prevented the shift in NBMPR sensitivity. In addition, inclusion of adenosine in the solubilization procedure increased the relative amount of NBMPR-resistant [3H]uridine uptake to 33 +/- 2% of the total influx in proteoliposomes prepared immediately after the proteins were extracted from the plasma membrane (i.e. no storage). A partial protection of [3H]NBMPR binding activity was also obtained using 2'-deoxyadenosine, 2-chloroadenosine, uridine, and non-radiolabelled NBMPR, but not with cytidine, inosine, diazepam, dipyridamole, or dilazep. These results suggest that both NBMPR sensitivity and transporter stability are dependent upon the conformational state of the protein. The protective effects of adenosine analogues and other nucleosides are likely due to their binding to the substrate translocation site, thereby effectively "locking" the transporter in a stable conformation.

Effect of detergents on ligand binding and translocation activities of solubilized/reconstituted nucleoside transporters.

Purification of equilibrative nucleoside transporters has been hampered by the functional instability of the detergent-solubilized proteins. A variety of nonionic detergents were compared with octylglucoside (the most commonly used detergent in this regard) for their abilities to solubilize functionally stable nucleoside transporter proteins from Ehrlich cell plasma membranes. Transporter stability was assessed through the binding of the specific probe [3H]nitrobenzylthioinosine to freshly solubilized and stored (48 h/6 degrees C) preparations. The most promising detergents were decylmaltoside and cyclohexylbutylmaltoside, both of which, like octylglucoside, solubilized over 70% of the transporters from the membrane. Decylmaltoside- and cyclohexylbutylmaltoside-solubilized transport proteins retained 61 and 83%, respectively, of their [3H]-nitrobenzylthioinosine binding activity upon storage, compared to about 30% using octylglucoside. Decylmaltoside was also superior to octylglucoside in its capacity to solubilize the transporter in a state that retained its high affinity for the transport inhibitors dilazep (Ki = 11 nM, vs 75 nM in octylglucoside) and dipyridamole (Ki = 260 nM, vs 12 microM in octylglucoside). Reconstitution studies indicated that both the decylmaltoside- and cyclohexylbutylmaltoside-solubilized transporters were capable of mediating the uptake of [3H]uridine. Decylmaltoside was superior to cyclohexylbutylmaltoside, however, in both the enhanced transport activity of the resulting proteoliposomes (Vi = 21 pmol/mg/s vs 13 pmol/mg/s, respectively) and the lower nonmediated uptake observed in the decylmaltoside-derived vesicles (27% of total uptake at 4 min incubation). Nevertheless, cyclohexylbutylmaltoside may be useful in initial solubilization procedures due to its ability to selectively solubilize the nucleoside transporter from the plasma membrane. The rational use of these detergents will enable a more extensive purification of functional nucleoside transporters.

Characterization of nucleoside transport activity in rabbit cortical synaptosomes.

Rabbit central nervous system (CNS) preparations have been used to study the central effects of adenosine, but little is known about the specific uptake mechanisms in rabbit brain involved in the regulation of extracellular adenosine concentrations. The present study assessed the kinetic and pharmacological characteristics of the uptake of [3H]uridine (a poorly metabolized substrate for adenosine transporters) by rabbit cortical synaptosomes, to define the transporter subtypes involved and to evaluate species variability in transporter characteristics. [3H]Uridine transport into rabbit cortical synaptosomes was mediated by two saturable, facilitated diffusion systems with characteristics compatible with the es and ei transporter subtypes identified in other mammalian species. About 65% of the total transport was mediated by the es system, and Km estimates of 320 and 94 microM were determined for [3H]uridine uptake by the es and ei transporter, respectively. These results differ significantly from the subtype ratio and kinetic characteristics reported for rat and guinea pig cortical synaptosomes, where most of the transport was mediated by an ei subtype. Dipyridamole, dilazep, nitrobenzylthioinosine, R75231, soluflazine, and mioflazine were relatively more effective as inhibitors of es-mediated uptake (compared with ei), while the substrates adenosine, cytidine, and guanosine did not distinguish between the es and ei transporters in rabbit cortical synaptosomes. These results highlight the significant species-tissue variability in nucleoside transporter characteristics and subtype expression, and emphasize the need to characterize the transporters in human CNS tissue to allow the rational development of CNS-active therapeutics based on inhibition of nucleoside transport.

Genetically-modified brewing yeasts for the 21st century. Progress to date.

Academic studies and traditional breeding of yeasts depend upon their sporulation lifestyle. The strains used have been specially selected to sporulate readily and to mate producing new yeast types. Unfortunately brewing yeast strains do not behave in this way. They sporulate poorly, any spores which are formed are usually non-viable and any haploid strains produced are invariably non-maters. Only in recent years, with the development of recombinant-DNA techniques, has the specific breeding of new brewing yeast strains become widespread. Strains have been produced with the ability to ferment a wider range of carbohydrates, with altered flocculation properties and which produce beers with modified flavours. Many have been tested on the pilot scale and one, an amylolytic brewing yeast, has received approval for commercial use.

Functional reconstitution of pharmacologically distinct subtypes of nucleoside transporters in liposomal membranes.

We examined the functional and pharmacological characteristics of liposome-reconstituted nucleoside transporter proteins obtained by detergent (octylglucoside) extraction from Ehrlich ascites tumor cell plasma membranes. Optimal reconstitution was achieved using a lipid composition of phosphatidylcholine, cholesterol, phosphatidylethanolamine and phosphatidylserine in a molar ratio of 33:33:26:8 and with a lipid-to-protein ratio of 30. This preparation had Km and Vmax values of 280 microM and 570 pmol/mg/sec, respectively, for the transporter-mediated uptake of [3H]uridine and bound 8.3 pmol of [3H]nitrobenzyl-thioinosine per milligram of protein. In general, the reconstituted system had kinetic and pharmacological characteristics comparable to those of the native membrane-located system, including an 80:20 ratio of nitrobenzylthioinosine-sensitive to -resistant [3H]uridine influx. The uridine translocation capacity of the optimally reconstituted system was 56 molecules per transporter per second compared with 104 molecules per transporter per second in intact cells, indicating that more than half of the inserted proteins were capable of mediating the influx of [3H]uridine (assuming that each functioning transporter was operating at optimal efficiency). Differences between the native and reconstituted transporters included the appearance of a [3H]uridine influx component (inhibited by adenosine) that was resistant to inhibition by R75231 (18%) and dipyridamole (10%). Dilazep was also significantly less effective in inhibiting the nitrobenzylthioinosine-sensitive transporter in the reconstituted preparations (IC50 = 41 nM) relative to that seen in intact Ehrlich cells (IC50 = 1.4 nM). These results suggest that inhibitor sensitivity may be a factor of the lipid microenvironment of the transporter or may involve other cellular components that could dissociate from the complex on detergent solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cellular differentiation on nucleoside transport in human neuroblastoma cells.

The nucleoside transport characteristics of undifferentiated and differentiated LA-N-2 human neuroblastoma cells were compared through measurement of the cellular accumulation of [3H]formycin B in the absence and presence of specific nucleoside transport blockers such as dipyridamole and nitrobenzylthioinosine (NBMPR). [3H]NBMPR was also used as a high affinity probe to obtain an estimate of the number of NBMPR-sensitive nucleoside transport proteins. Undifferentiated LA-N-2 cells accumulated [3H]formycin B (25 microM) via a NBMPR/dipyridamole sensitive, Na(+)-independent, nucleoside transport system (Vi = 1.52 pmol/microliters/s; maximum intracellular concentration = 45 pmol/microliters cell water). The undifferentiated cells also had a high density of site-specific [3H]NBMPR binding sites (135,000 sites/cell; KD = 0.4 nM). When cell differentiation was induced by exposure to a serum-free defined medium, the initial rate of transporter-mediated [3H]formycin B uptake increased to 1.92 pmol/microliters/s, and the steady-state intracellular concentration of [3H]formycin B also increased significantly to 73 pmol/microliters. However, there was no concomitant change in the number of [3H]NBMPR binding sites, and the additional uptake was not Na(+)-dependent. This enhanced uptake in the differentiated cells appeared to be due, in part, to an increased functional expression of a NBMPR-resistant form of facilitated nucleoside transporter. Approximately 18% of the transporter-mediated uptake in the differentiated cells was resistant to inhibition by NBMPR at concentrations that blocked transport completely in the undifferentiated cells. This cell model may prove useful for basic studies on regulation of nucleoside transporter subtype expression in neural tissues, and for evaluation of the efficacy and potential host toxicity of cytotoxic nucleoside analogues (+/- specific transport blockers) in the treatment of neuroblastoma.

Biochemical and Molecular Characterization of Obesumbacterium proteus, a Common Contaminant of Brewing Yeasts.

We have evaluated the effectiveness of API 20E, Biolog testing, plasmid profiling, ribotyping, and enteric repetitive intergenic consensus (ERIC)-PCR to characterize, classify, and differentiate nine bacterial isolates of the common brewery contaminant Obesumbacterium proteus. Of the five typing techniques, Biolog testing, plasmid profiling, and ERIC-PCR provided the most differentiation, and API 20E testing and ribotyping were relatively indiscriminate. The molecular biology approach of ERIC-PCR offered the ideal combination of speed, simplicity, and discrimination in this study. Overall, the results are supportive of the view that O. proteus can be subdivided into two biogroups, biogroup 1, which has considerable biochemical and genetic homology to Hafnia alvei, and biogroup 2, which is relatively heterogeneous.

Interaction of the mioflazine derivative R75231 with the nucleoside transporter: evidence for positive cooperativity.

This study investigated the interaction of the mioflazine derivative R75231 with the nucleoside transport system of rabbit cortical synaptosomes, and assessed the binding of [3H]R75231 to human erythrocyte ghost membranes. R75231 was a potent inhibitor of [3H]nitrobenzylthioinosine binding and [3H]uridine uptake in synaptosomes (Ki < 10 nM). This inhibition was evident even after extensive washing of the synaptosomes, subsequent to exposure to R75231. In addition to its tight binding characteristics, R75231 was shown to be a 'mixed' type inhibitor of [3H]nitrobenzylthioinosine binding (increased KD, decreased Bmax). [3H]R75231 bound with high affinity (KD = 0.4 nM) to erythrocyte membranes with a Bmax of 44 pmol/mg protein, which is comparable to the number of [3H]nitrobenzylthioinosine binding sites in this preparation. Binding of [3H]R75231 to these membranes was reversible, but the rate of dissociation was dependent upon the displacer used. Nitrobenzylthioinosine and dipyridamole each induced a complete dissociation of site-bound [3H]R75231 at rates not significantly different from those observed using a protocol involving a 100-fold dilution with buffer (no displacer). However, R75231 and mioflazine slowed the rate of dissociation of [3H]R75231 and actually caused an initial increase in the amount of site-bound [3H]R75231. Adenosine, on the other hand, enhanced the rate of [3H]R75231 dissociation. These results indicate that R75231 binding to the nucleoside transporter is a complex reaction, which may involve multiple interacting sites demonstrating positive cooperativity.

Differential uptake of [3H]guanosine by nucleoside transporter subtypes in Ehrlich ascites tumour cells.

Intracellular metabolism of [3H]guanosine was minimal (< 15%) during the first 22 s of incubation, and hence reasonable estimates of initial-rate influx kinetics could be derived by using metabolically active cells. Na(+)-dependent concentrative [3H]guanosine uptake was not observed. Data suggest that [3H]guanosine was accumulated primarily via the nitrobenzylthioguanosine (NBTGR)-sensitive subtype of facilitated nucleoside transporter. Incubation of cells with 100 nM-NBTGR significantly decreased the potency of guanosine as an inhibitor of [3H]uridine influx. The Vmax. for [3H]guanosine influx (9.2 pmol/s per microliters) was significantly lower than that for [3H]uridine influx (16 pmol/s per microliters). The Km for transporter-mediated [3H]guanosine influx determined in the presence of 100 nM-NBTGR was 16-fold higher (1780 microM) than that determined in its absence, whereas the Km for [3H]uridine influx was shifted by only 2-fold. In other respects, the cellular accumulations of [3H]guanosine and [3H]uridine were similar; both had Km values of approx. 140 microM for total mediated influx, and both were inhibited similarly by other nucleosides and transport inhibitors. These characteristics, and the fact that guanosine is an endogenous nucleoside, suggest that [3H]guanosine may prove useful as a poorly metabolized, relatively selective, substrate for study of the NBTGR-sensitive nucleoside transport systems of mammalian cells.

Heterogeneity of [3H]dipyridamole binding to CNS membranes: correlation with [3H]nitrobenzylthioinosine binding and [3H]uridine influx studies.

The relationship between the nucleoside transport system and the nitrobenzylthioinosine-sensitive and -resistant [3H]dipyridamole binding sites was examined by comparing the characteristics of [3H]dipyridamole binding with those of [3H]nitrobenzylthioinosine binding and [3H]-uridine influx in rabbit and guinea pig cerebral cortical synaptosomes. Two distinct high-affinity synaptosomal membrane-associated [3H]dipyridamole binding sites, with different sensitivities to inhibition by nitrobenzylthioinosine, were characterized in the presence of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, 0.01%) to prevent [3H]dipyridamole binding to glass tubes and filters. The nitrobenzylthioinosine-resistant [3H]-dipyridamole binding sites represented a greater proportion of the total membrane sites in guinea pig than in rabbit (40 vs. 10% based on inhibition studies). In rabbit, nitrobenzylthioinosine-sensitive [3H]dipyridamole binding (KD = 1.4 +/- 0.2 nM) and [3H]nitrobenzylthioinosine binding (KD = 0.30 +/- 0.01 nM) appeared to involve the same membrane site associated with the nitrobenzylthioinosine-sensitive nucleoside transporter. By mass law analysis, [3H]-dipyridamole binding in guinea pig could be resolved into two components based on sensitivity to inhibition by 1 microM nitrobenzylthioinosine. The nitrobenzylthioinosine-resistant [3H]dipyridamole binding sites were relatively insensitive to inhibition by all of the nucleoside transport substrates and inhibitors tested, with the exception of dipyridamole itself. In guinea pig synaptosomes, 100 microM dilazep blocked nitrobenzylthioinosine-resistant [3H]uridine transport completely but inhibited the nitrobenzylthioinosine-resistant [3H]dipyridamole binding component by only 20%. Furthermore, a greater percentage of the [3H]dipyridamole binding was nitrobenzylthioinosine resistant in guinea pig compared with rabbit, yet both species had a similar percentage of nitrobenzylthioinosine-resistant [3H]uridine transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative pharmacology of the nitrobenzylthioguanosine-sensitive and -resistant nucleoside transport mechanisms of Ehrlich ascites tumor cells.

A variety of nucleoside transport inhibitors and substrates were compared for their capacities to inhibit the zero-trans influx of [3H]uridine in Ehrlich ascites tumor cells. ATP-depleted cells accumulated [3H]uridine primarily by facilitated diffusion (Vmax = 16 pmol/sec/microliter cell water) via both nitrobenzylthioguanosine (NBTGR)-sensitive (IC50 = 0.53 nM, 100 microM [3H]uridine) and NBTGR-resistant (IC50 = 71 microM, 100 microM [3H]uridine) mechanisms with uridine Km estimates of 99 and 284 microM, respectively. Dilazep also distinguished between the transporter subtypes with IC50 values of 1.4 nM and 1.8 microM, respectively, for inhibiting 100 microM [3H]uridine influx. Incubation of cells with 50 nM NBTGR allowed the selective study of inhibitor effects on NBTGR-resistant [3H]uridine influx. Dipyridamole, cyclopentyladenosine, 2-phenylaminoadenosine, etoposide, teniposide, diazepam, chlordiazepoxide, triazolam and the lidoflazine derivative 2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis-(4- fluorophenyl)pentyl]-1-piperazineacetamide (R75231), were significantly less potent as inhibitors of NBTGR-resistant influx, when compared with their capacities to inhibit the total mediated influx of [3H]uridine. In contrast, 2-fluoroadenosine, 2-chloroadenosine, 5'-N-ethylcarboxamidoadenosine and soluflazine were relatively more effective as inhibitors of the NBTGR-resistant component. Mioflazine, a compound related to both soluflazine and R75231, did not distinguish between transporter subtypes. The NBTGR-resistant transporter also had a distinctive substrate specificity; guanosine, 2'-deoxyguanosine, cytidine and 2'-deoxycytidine were significantly less effective as inhibitors of NBTGR-resistant [3H]uridine influx.(ABSTRACT TRUNCATED AT 250 WORDS)