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.

Enhanced efflux of [3H]vinblastine from Chinese hamster ovary cells transfected with a full-length complementary DNA clone for the mdr1 gene.

Multidrug-resistant Chinese hamster ovary cell clones stably transfected with, and overexpressing, the mouse mdr1 complementary DNA clone along with drug-sensitive Chinese hamster ovary control cells were characterized for their capacities to accumulate and retain [3H]vinblastine. Multidrug-resistant mdr1 transfectants show a 3-4-fold decrease in [3H]vinblastine accumulation, compared to their drug-sensitive counterparts. After ATP depletion, this difference in [3H]vinblastine accumulation between mdr1 transfectants and control cells effectively disappears. This ATP-dependent decreased drug accumulation is paralleled in mdr1 transfectants by an enhanced capacity of these cells to extrude the drug in an ATP-dependent manner. In medium containing glucose and glutamine, the mdr1 transfectants release preloaded drug at a rate five times that of control, drug-sensitive cells. In ATP-depleted control and mdr1-transfected cells, there is little difference in the rate or extent of [3H]vinblastine release. The observation that the mdr1 transfectants show a decreased [3H]vinblastine accumulation and an increased vinblastine release, both of which are abolished when cellular ATP levels are reduced, provides a direct demonstration that the product of the transfected mdr1 gene is responsible for a mechanism controlling cellular drug levels in an ATP-dependent manner. However, attempts to establish competition for [3H]vinblastine transport by vincristine, daunomycin, and actinomycin D were only partly successful in mdr1 transfectants.

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.

Benzodiazepine inhibition of nucleoside transport in human erythrocytes.

The interaction of several benzodiazepines (BDZs) with the nucleoside transport system of fresh erythrocytes from humans was investigated. The affinities of BDZs for the nucleoside transport system were estimated by measuring BDZ inhibition of (a) the site-specific binding of nitrobenzylthioinosine, a potent and specific inhibitor of nucleoside transport, and (b) the uridine transport processes, zero-trans influx, zero-trans efflux, and equilibrium exchange influx. The BDZs inhibited both the inward and outward transport processes, and, for individual agents, inhibition constants (Ki) were similar for the inhibition of each transport process and for the inhibition of the site-specific binding of nitrobenzylthioinosine. The order of potencies of the BDZs in their interactions with the nucleoside transport mechanism (Ro 5-4864 greater than diazepam greater than clonazepam greater than lorazepam greater than flurazepam) is distinct from the potencies of these compounds at BDZ recognition sites. The affinities of the BDZs for the nucleoside transport system, which are about 1000-fold lower than for BDZ recognition sites, suggest that significant inhibition is unlikely to occur with the plasma concentrations (less than 1 microM) that result from usual anxiolytic doses of these agents.

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.

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.

Modulation of [3H]flunitrazepam binding to rat cerebellar benzodiazepine receptors by phosphatidylserine.

The influence of phosphatidylserine on ligand binding to the benzodiazepine/GABA receptor complex was assessed in rat cerebellar synaptic membranes and in a detergent-solubilized membrane preparation. Intact synaptic membranes or membranes solubilized with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate) were incubated with a range of concentrations of phosphatidylserine for 2 h at 4 degrees C, prior to use in radioligand binding assays. Phosphatidylserine, an endogenous membrane phospholipid, facilitated the site-specific binding of [3H]flunitrazepam to synaptic membranes and CHAPS-solubilized preparations. In addition, phosphatidylserine inhibited the facilitation of [3H]flunitrazepam binding induced by either cartazolate or gamma-aminobutyric acid (GABA). Although the maximum effect (38% facilitation of [3H]flunitrazepam binding; greater than 90% inhibition of the cartazolate action) was produced using 130 microM phosphatidylserine, a significant enhancement of [3H]flunitrazepam binding could be observed upon preincubation of synaptic membranes with concentrations of phosphatidylserine as low as 5 microM. These results suggest that endogenous phosphatidylserine may play a role in the regulation of benzodiazepine/GABA receptor function, possibly through modulation of the mechanisms which functionally link the various components of this complex receptor system.

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.

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.

Kinetic analysis of ligand binding to the Ehrlich cell nucleoside transporter: pharmacological characterization of allosteric interactions with the [3H]nitrobenzylthioinosine binding site.

Kinetic analysis of the binding of [3H]nitrobenzylthioinosine ([3H] NBMPR) to Ehrlich ascites tumor cell plasma membranes was conducted in the presence and absence of a variety of nucleoside transport inhibitors and substrates. The association of [3H] NBMPR with Ehrlich cell membranes occurred in two distinct phases, possibly reflecting functional conformation changes in the [3H]NBMPR binding site/nucleoside transporter complex. Inhibitors of the equilibrium binding of [3H]NBMPR, tested at submaximal inhibitory concentrations, generally decreased the rate of association of [3H]NBMPR, but the magnitude of this effect varied significantly with the agent tested. Adenosine and diazepam had relatively minor effects on the association rate, whereas dipyridamole and mioflazine slowed the rate dramatically. Inhibitors of nucleoside transport also decreased the rate of dissociation of [3H]NBMPR, with an order of potency significantly different from their relative potencies as inhibitors of the equilibrium binding of [3H]NBMPR. Dilazep, dipyridamole, and mioflazine were effective inhibitors of both [3H]NBMPR dissociation and equilibrium binding. The lidoflazine analogue R75231, on the other hand, had no effect on the rate of dissociation of [3H]NBMPR at concentrations below 300 microM, even though it was one of the most potent inhibitors of [3H]NBMPR binding tested (Ki less than 100 nM). In contrast, a series of natural substrates for the nucleoside transport system enhanced the rate of dissociation of [3H]NBMPR with an order of effectiveness that paralleled their relative affinities for the permeant site of the transporter. The most effective enhancers of [3H]NBMPR dissociation, however, were the benzodiazepines diazepam, chlordiazepoxide, and triazolam. Comparable effects of adenosine and dipyridamole on [3H]NBMPR dissociation rate were obtained upon solubilization of the membranes with octylglucoside, suggesting that this phenomenon was not due to changes in membrane fluidity. These results are compatible with the existence of specific ligand recognition sites on the nucleoside transport complex of Ehrlich cells that are pharmacologically distinct from, but allosterically linked to, the high affinity binding sites for [3H]NBMPR. The marked effects on [3H]NBMPR binding kinetics that result from ligand interactions with these sites must be considered in the design and analysis of all studies involving the use of [3H]NBMPR as a high affinity probe for the nucleoside transport system.

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.

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)

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)

Photoaffinity labelling of benzodiazepine receptors: lack of effect on ligand binding to the nucleoside transport system.

This study was undertaken to investigate the possibility of an allosteric interaction between benzodiazepine receptors and the CNS nucleoside transport system. Irreversible (photoaffinity) labelling of the benzodiazepine receptors in guinea pig cortical membranes resulted in a marked reduction in the binding (Bmax) of both [3H]flunitrazepam (71%) and [3H]ethyl-beta-carboline-3-carboxylate (22%) to the benzodiazepine receptors but had no effect on the binding of [3H]nitrobenzylthioinosine to the nucleoside transport system. Furthermore, although photoaffinity labelling resulted in a significant decrease in the affinities of flunitrazepam (approximately equal to 16-fold) and dipyridamole (approximately equal to sevenfold) for the [3H]Ro 15-1788 binding site of the benzodiazepine receptor complex, the affinities of these compounds for the nucleoside transport system were unaltered. These results suggest that the CNS nucleoside transport system and the benzodiazepine receptor complex are distinct, noninteractive ligand recognition sites.