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S M Jarvis

Publications and source records attributed to S M Jarvis.

At least 55 records · Page 3Linked to original sources
PubMed1991

Expression of the rabbit intestinal N2 Na+/nucleoside transporter in Xenopus laevis oocytes.

Polyadenylated [poly(A)+] mRNA isolated from rabbit small-intestinal mucosa was injected into Xenopus laevis oocytes, and expression of the N2 Na+/nucleoside co-transporter was assayed by measuring Na(+)-dependent thymidine uptake. Expression of Na(+)-dependent thymidine uptake steadily increased after mRNA injection and was on average increased 11-fold by day 6 over background. Na(+)-dependent thymidine uptake was saturable (apparent Km approximately 30 microM at 22 degrees C) and inhibited by uridine and cytidine, but not by guanosine and inosine. These properties of the expressed thymidine transport strongly suggest that the epithelial N2 Na+/nucleoside co-transporter can be expressed in X. laevis oocytes.

Animals↗
PubMed1991

Multiple sodium-dependent nucleoside transport systems in bovine renal brush-border membrane vesicles.

Na(+)-dependent nucleoside transport was examined in bovine renal brush-border membrane vesicles. Two separate Na+/nucleoside cotransporters were shown to be present: (1) a system specific for purine nucleosides and uridine, designated as the N1 carrier, and (2) an Na(+)-dependent nucleoside transporter that accepts pyrimidine nucleosides, adenosine and analogues of adenosine, designated as the N2 system. Both systems exhibit a high affinity for nucleosides (apparent Km values approximately 10 microM), are insensitive to inhibition by facilitated-diffusion nucleoside transport inhibitors, are rheogenic and exhibit a high specificity for Na+. Na+ increases the affinity of the influx of guanosine and thymidine, nucleosides that serve as model permeants for the N1 and N2 nucleoside transporters respectively. The Na+/nucleoside coupling stoichiometry is consistent with 1:1 for both carriers.

Animals↗
PubMed1991

Adenosine transport and nitrobenzylthioinosine binding in human placental membrane vesicles from brush-border and basal sides of the trophoblast.

The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2'-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2'-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparent Km approx. 150 microM) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparent Kd 0.98 +/- 0.21 nM). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparent Ki values were 0.23 +/- 0.012, 0.36 +/- 0.035, 0.78 +/- 0.1, 0.70 +/- 0.12 (mM), and 0.12 and 4.2 +/- 1.4 (nM). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brush-border membrane vesicles (apparent Kd 1.05 +/- 0.13 nM and apparent Ki values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14 +/- 0.045, 0.54 +/- 0.046, 1.26 +/- 0.20, 1.09 +/- 0.18 mM and 0.14 and 3.7 +/- 0.5 nM, respectively). Exposure of both membrane vesicles to UV light in the presence of [3H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparent Mr on SDS gel electropherograms of 77,000-45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surfaces of the human placental syncytiotrophoblast possess broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters.

Adenosine↗
PubMed1990

Differential inhibition of nucleoside transport systems in mammalian cells by a new series of compounds related to lidoflazine and mioflazine.

The sensitivity of facilitated-diffusion and Na(+)-dependent nucleoside transporters to inhibition by a series of novel compounds related to lidoflazine and mioflazine was investigated. Uridine transport by rabbit erythrocytes, which proceeds solely by the nitrobenzylthioinosine (NBMPR)-sensitive facilitated-diffusion system, was inhibited with apparent Ki values of less than 10 nM by lidoflazine, mioflazine, soluflazine and R73-335. These compounds also blocked site-specific [3H]NBMPR binding to rabbit erthrocyte membranes in a competitive fashion. The NBMPR-sensitive system in rat erythrocytes was also inhibited by lidoflazine, mioflazine, soluflazine and R73-335 but was two to three orders of magnitude less sensitive to inhibition than the system in rabbit erythrocytes (apparent Ki 7.3, 2.4, 5.7 and 0.1 microM, respectively). Lidoflazine, mioflazine and R73-335 exhibited a similar potency for the NBMPR-sensitive and -insensitive nucleoside transporters in rat erythrocytes. In contrast, soluflazine was 20- to 100-fold more potent as an inhibitor of the NBMPR-insensitive nucleoside transport component in rat erythrocytes (IC50 of 0.08-0.2 microM) compared to the NBMPR-sensitive nucleoside carrier in these cells (IC50 approximately 10 microM). None of the test compounds were potent inhibits of Na(+)-dependent uridine transport in bovine renal brush-border membrane vesicles. These results indicate that lidoflazine, mioflazine, soluflazine and R73-335 are selective inhibitors of nucleoside transport in animal cells and that the potency of these compounds as nucleoside transport inhibitors is species dependent.

Affinity Labels↗
PubMed1990

Transport characteristics of renal brush border Na(+)- and K(+)-dependent uridine carriers.

The uptake of uridine into rat renal brush-border membrane vesicles is mediated by Na(+)- and K(+)-dependent concentrative transport processes. At a 100 mM extravesicular cation concentration the apparent Km values were 9.7 +/- 4.2 and 28 +/- 5 microM, and Vmax values were 28 +/- 4 and 7 +/- 1 pmol.mg protein-1.s-1 for the Na(+)- and K(+)-dependent systems, respectively. Uracil, D-ribose, and D-glucose failed to inhibit the uptake processes, indicating that these carriers are specific for nucleosides. Other purines and pyrimidines inhibited uridine uptake competitively, although these two transport systems seem to favor adenosine and pyrimidines as permeants. Evidence is also given that transport is rheogenic, involving a net transfer of positive charge. The Na+:uridine and K+:uridine coupling stoichiometry was found to be 1:1 and 3:2, respectively. Both systems can also be driven by an anion gradient with apparent NO3- affinity (KNO3-) values of 42 +/- 13 and 163 +/- 54 mM for Na(+)- and K(+)-dependent systems, respectively.

Animals↗
PubMed1989

Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.

Animals↗
PubMed1989

Characterization of sodium-dependent nucleoside transport in rabbit intestinal brush-border membrane vesicles.

The characteristics of uridine transport were studied in rabbit intestinal brush-border membrane vesicles. Uridine was taken up into an osmotically active space in the absence of metabolism and there was no binding of uridine to the membrane vesicles. Uridine uptake was markedly enhanced by sodium, but showed no significant stimulation by other monovalent cations tested. Kinetic analysis of the sodium-dependent component of uridine flux indicated a single system obeying Michaelis-Menten kinetics (Km value of 6.4 +/- 1.4 microM with a Vmax of 9.1 +/- 3.6 pmol/mg protein per s as measured under zero-trans conditions with a 100 mM NaCl gradient at 24 degrees C). A variety of purine and pyrimidine nucleosides were able to inhibit sodium-dependent uridine transport, suggesting that these nucleosides are also permeants for the same system. Consistent with this suggestion was the finding that these nucleosides also stimulated uridine efflux from the brush-border membrane vesicles. The sodium: uridine coupling stoichiometry was found to be 1:1 as measured by the activation method. From these results it is concluded that a broad specificity sodium-dependent nucleoside transporter is present at the brush-border membrane surface of rabbit enterocytes.

Animals↗
PubMed1988

Na+- and K+-dependent uridine transport in rat renal brush-border membrane vesicles.

The transport of uridine into rat renal brush-border membrane vesicles was investigated using an inhibitor-stop filtration method. Uridine was not metabolized under these conditions. The rapid efflux of intravesicular uridine was prevented by adding 1 mM phloridzin to the ice-cold stop solution. In the presence of inwardly directed gradients of either Na+ or K+, zero-trans uridine uptake exhibited a transient overshoot phenomenon indicating active transport. The overshoot was much more pronounced with Na+ than K+ and it was not observed when either Na+ or K+ was at equilibrium across the membrane. The K+-induced overshoot was not due to the presence of a membrane potential alone, as an inwardly directed gradient of choline chloride failed to produce it. The amplitude of the overshoot was increased by raising either the Na+ or K+ concentration outside the membrane or by using more lipophilic anions (reactive order was NO3- greater than SCN- greater than Cl- greater than SO4(2-). Zero-trans efflux studies showed that the uridine transport is bidirectional. Li+ could substitute poorly for Na+ but not at all for K+. Stoichiometries of 1:1 and greater than 1:1 were observed for Na+: uridine and K+: uridine coupling, respectively. A preliminary analysis of the interactions between Na+ and K+ for uridine uptake showed complex interactions which can best be explained by the involvement of two different systems for nucleoside transport in the rat renal brush-border membrane, one requiring Na+ and the other K+ as transport coupler.

Animals↗
PubMed1988

Inhibition by nucleosides of glucose-transport activity in human erythrocytes.

The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glucose-sensitive cytochalasin B binding in an apparently competitive manner. Thymidine, a nucleoside not metabolized by human erythrocytes, inhibited glucose influx by intact cells with an IC50 value of 9 mM when preincubated with the erythrocytes. In contrast, thymidine was an order of magnitude less potent as an inhibitor of glucose influx when added simultaneously with the radioactive glucose. Consistent with this finding was the demonstration that glucose influx by inside-out vesicles prepared from human erythrocytes was more susceptible to thymidine inhibition than glucose influx by right-side-out vesicles. These data, together with previous suggestions that cytochalasin B binds to the glucose carrier at the inner face of the membrane, indicate that nucleosides are capable of inhibiting glucose-transport activity by interacting at the cytoplasmic surface of the glucose transporter. Nucleosides may also exhibit a low-affinity interaction at the extracellular face of the glucose transporter.

Biological Transport↗
PubMed1988

Nucleoside transport in rat cerebral-cortical synaptosomes. Evidence for two types of nucleoside transporters.

The transport of [U-14C]uridine was investigated in rat cerebral-cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions the rapid efflux of uridine from the synaptosomes is prevented and uridine is not significantly metabolized in the synaptosome during the first 1 min of uptake. The dose-response curve for the inhibition of uridine transport by nitrobenzylthioinosine (NBMPR) was biphasic: approx. 40% of the transport activity was inhibited with an IC50 (concentration causing half-maximal inhibition) value of 0.5 nM, but the remaining activity was insensitive to concentrations as high as 1 microM. Similar biphasic dose-response curves were observed for dilazep inhibition, but both transport components were equally sensitive to dipyridamole inhibition. Uridine influx by both components was saturable (Km 300 +/- 51 and 214 +/- 23 microM, and Vmax. 12 +/- 3 and 16 +/- 3 pmol/s per mg of protein, for NBMPR-sensitive and NBMPR-insensitive components respectively), and inhibited by other nucleosides such as 2-chloroadenosine, adenosine, inosine, thymidine and guanosine with similar IC50 values for the two components. Inhibition of uridine transport by NBMPR was associated with high-affinity binding of NBMPR to the synaptosome membrane (Kd 58 +/- 15 pM). Binding of NBMPR to these sites was competitively blocked by uridine and adenosine and inhibited by dilazep and dipyridamole, with Ki values similar to those measured for inhibiting NBMPR-sensitive uridine influx. These results demonstrate that there are two components of nucleoside transport in our rat synaptosomal preparation that differ in their sensitivity to inhibition by NBMPR. Thus conclusions regarding nucleoside transport in rat brain based only on NBMPR-binding activity must be viewed with caution.

Animals↗
PubMed1988

Identification of glucose and nucleoside transport proteins in neonatal pig erythrocytes using monoclonal antibodies against band 4.5 polypeptides of adult human and pig erythrocytes.

Cytochalasin B and nitrobenzylthioinosine (NBMPR), which inhibit membrane transport of glucose and nucleosides, respectively, have served as photoaffinity ligands that become covalently linked at inhibitor binding sites on transporter-associated proteins. Thus, when membranes from erythrocytes of neonatal pigs with site-bound [3H]cytochalasin B or [3H]NBMPR were irradiated with uv light, two labeled membrane polypeptides (peak Mr values: 55,000 and 64,000, respectively) were identified. Treatment of the photolabeled membranes with endoglycosidase F increased the mobility of [3H]cytochalasin B- and [3H]NBMPR-labeled material (peak Mr values: 44,000 and 57,000, respectively) and limited digestion with trypsin yielded different polypeptide fragments (Mr values: 18,000-23,000 and 43,000, respectively). Identification of the photolabeled polypeptides as transporter components was established using monoclonal antibodies (MAbs) raised against partially purified preparations of band 4.5 from erythrocytes of adult pigs and humans. MAbs 65D4 and 64C7 (anti-human band 4.5), raised in this study, reacted with [3H]cytochalasin B-labeled material from membranes of human erythrocytes and bound to permeabilized erythrocytes but not to intact cells. MAb 65D4 also bound to erythrocytes of mice and neonatal pigs and to a variety of cultured cells (mouse, human, rat), including AE1 mouse lymphoma cells, which lack an NBMPR-sensitive nucleoside transporter. Also employed was MAb 11C4 (anti-pig band 4.5), which recognizes the NBMPR-binding protein of erythrocyte membranes from adult pigs. When membrane proteins from neonatal and adult pigs were subjected to electrophoretic analysis and blots were probed with different MAbs, MAb 65D4 (anti-human band 4.5) bound to material that comigrated with [3H]cytochalasin B-labeled polypeptides (band 4.5) from neonatal, but not adult, pig erythrocytes, whereas MAb 11C4 (anti-pig band 4.5) bound to material that comigrated with [3H]NBMPR-labeled band 4.5 polypeptides of erythrocytes from both neonatal and adult pigs. These results, which indicate structural differences in the cytochalasin B- and NBMPR-binding proteins of pig erythrocytes, establish the presence of both proteins in erythrocytes of neonatal pigs and suggest that only the NBMPR-binding protein is present in erythrocytes of adult pigs.

Affinity Labels↗
PubMed1987

Purification and reconstitution studies of the nucleoside transporter from pig erythrocytes.

The pig erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 64,000) on the basis of photoaffinity labelling experiments with the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). This protein was purified 140-fold by treatment of haemoglobin-free erythrocytes 'ghosts' with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with n-octyl-glucoside and subsequent gradient-elution ion-exchange chromatography on DEAE-cellulose. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified material revealed the presence of only two detectable protein bands, one which co-migrated with the radiolabelled NBMPR-binding protein, and a lower molecular weight species with an Mr of 43,000. The latter protein may be a degradation product of the band 3 anion-exchange transporter. The overall purification of the NBMPR-binding protein with respect to the Mr 64,000 band was 350-fold. Reversible NBMPR-binding to the partially-purified band 4.5 preparation was saturable (apparent Kd 7.2 nM). Adjustment of the chromatography conditions to allow elution of the NBMPR-binding protein along with the majority of solubilised membrane phospholipid reduced the apparent Kd value to 3.0 nM. Purification of reversible NBMPR-binding activity during ion-exchange chromatography was paralleled by an increase in the specific activity of nitrobenzylthioguanosine (NBTGR) -sensitive uridine transport as assayed in proteoliposomes reconstituted by a freeze-thaw-sonication procedure.

Affinity Labels↗