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S M Jarvis

Publications and source records attributed to S M Jarvis.

At least 37 records · Page 2Linked to original sources
PubMed1995

Immunolocalisation of nucleoside transporters in human placental trophoblast and endothelial cells: evidence for multiple transporter isoforms.

Polyclonal antibodies raised against the human erythrocyte nucleoside transporter were used to investigate the distribution of the nucleoside transporters in the placenta. Immunoblots of brush-border membranes isolated from the human syncytiotrophoblast revealed a cross-reactive species that co-migrated with the erythrocyte nucleoside transporter as a broad band of apparent M(r) 55,000. In contrast, no labelling was detected in basal membranes containing a similar number of equilibrative nucleoside transporters as assessed by nitrobenzylthioinosine (NBMPR)-binding. The absence of cross-reactive epitopes in basal membranes and their presence in brush-border membranes was confirmed by confocal immunofluorescence microscopy. These results suggest that at least two isoforms of the NBMPR-sensitive nucleoside transporter are present in the human placenta. The lumenal surfaces of fetal capillaries, small placental vessels and umbilical vein were also strongly labelled by the antibody, a finding that suggests that the high fetal-placental adenosine uptake previously reported is due to endothelial transporters.

Affinity Labels↗
PubMed1995

Quantitation and immunolocalization of glucose transporters in the human placenta.

The subcellular distributions of the mammalian passive glucose transporter isoforms GLUT1, GLUT3 and GLUT4, in the human placenta, were investigated using isoform-specific anti-peptide antibodies. On western blots of both basal and brush-border plasma membranes isolated from the syncytiotrophoblast, antibodies specific for GLUT1 labelled a broad band (apparent Mr 55,000) that co-migrated with the human erythrocyte GLUT1 glucose transporter. In contrast, no labelling was detectable when blots were probed with antibodies specific for the GLUT3 or GLUT4 isoforms. Densitometric analysis of blots showed that GLUT1 accounts for approximately 90 and 65 per cent of the D-glucose-sensitive cytochalasin B binding sites present in brush-border and basal membranes, respectively. Confocal immunofluorescence microscopy of fixed placental tissue showed that GLUT1 is abundant at both maternal- and fetal-facing surfaces of the syncytiotrophoblast whereas it was undetectable at the fetal capillary endothelium. In parallel experiments, no staining by antibodies against either the GLUT3 or the GLUT4 isoforms was detected in placental tissue. These results indicate that GLUT1 is the major isoform responsible for glucose transfer from mother to fetus. The absence of GLUT4 is consistent with the lack of insulin-sensitive glucose transport across the placenta.

Erythrocyte Membrane↗
PubMed1995

Transport and metabolism of adenosine in diabetic human placenta.

Pregnancy complicated by diabetes is a relatively frequent event and may result in fetal embriopathy. However, little is known regarding whether placental transport functions are altered. In this study, we have compared the activity of the nitrobenzylthioinosine (NBMPR)-sensitive adenosine transporter and adenosine metabolism in human placental brush-border- and basal-membrane vesicles from placentas of normal and diabetic pregnancies. Neither [3H]NBMPR binding, a marker of the facilitative-diffusion nucleoside transporter in the human placenta, nor adenosine metabolism exhibited a significant difference in either the brush-border- or the basal-membrane vesicles between the normal and diabetic group, except for an increased affinity in [3H]NBMPR binding at the basal side in diabetic placenta. This result contrasts with an earlier finding using the same group of patients that adenosine transport is downregulated in umbilical vein endothelial cells from diabetic pregnancies. It is concluded that adenosine transport is modulated selectively in different tissues in diabetic pregnancies.

Adenosine↗
PubMed1994

Characterization of a sodium-dependent concentrative nucleobase-transport system in guinea-pig kidney cortex brush-border membrane vesicles.

The characteristics of hypoxanthine transport were examined in purified brush-border membrane vesicles isolated from guinea-pig kidney. Hypoxanthine uptake in the vesicles was specifically stimulated by both Na+ and an inside-negative potential, resulting in a transient accumulation of intravesicular hypoxanthine. Na(+)-dependent hypoxanthine influx was saturable (apparent Km 4.4 +/- 2.1 microM, Vmax. 128 +/- 29 pmol/min per mg of protein at 100 mM NaCl and 22 degrees C). Guanine, thymine, 5-fluorouracil and uracil inhibited hypoxanthine uptake (Ki values 1-30 microM), but adenine and the nucleosides inosine and thymidine were without effect. Guanine competitively inhibited Na(+)-dependent hypoxanthine influx, suggesting that it was a substrate for the active nucleobase transporter in guinea-pig renal membrane vesicles. A sigmoidal dependence between hypoxanthine influx and Na+ concentration was obtained (KNa 13 +/- 2 mM; Hill coefficient, h, 2.13 +/- 0.14), suggesting that at least two Na+ ions are transported per hypoxanthine molecule. This system differs from the Na(+)-nucleobase carrier in cultured LLC-PK1 renal cells, which has a stoichiometric coupling ratio of 1:1. These results represent the first demonstration of an active electrogenic nucleobase carrier in renal apical membrane vesicles.

Animals↗
PubMed1994

Nucleoside influx and efflux in guinea-pig ventricular myocytes. Inhibition by analogues of lidoflazine.

Adenosine influx and formycin B influx and efflux were characterized in guinea-pig ventricular myocytes at 22 degrees. Transport by both modes was saturable and inhibited by nitrobenzylthioinosine (NBMPR), indicating the presence of an equilibrative NBMPR-sensitive nucleoside transporter in the cardiomyocytes. The kinetic constants for influx and efflux of formycin B, a non-metabolized nucleoside, were similar, suggesting that the nucleoside transporter exhibits symmetrical kinetics (apparent Km 490 +/- 160 and 700 +/- 140 microM; Vmax 6.5 +/- 1.7 and 3.5 +/- 0.3 nmol/10(6) cells per min for influx and efflux, respectively). No evidence was found of either NBMPR-insensitive equilibrative nucleoside transport or sodium-dependent concentrative nucleoside transport. Inhibition of adenosine influx (apparent Km100 +/- 33 microM), by lidoflazine and the analogues mioflazine, soluflazine and R73-335, gave average Ki values of 730, 100, 64 and 2.9 nM, respectively. These compounds also inhibited formycin B efflux with a similar potency to that of adenosine influx. NBMPR-sensitive nucleoside transport was associated with high affinity binding of NBMPR (apparent Kd approximately 1 nM; 9.6 x 10(5) sites/cell). Specific binding of NBMPR was also inhibited by lidoflazine and its analogues. Mioflazine and soluflazine were 20-30-fold more potent at inhibiting NBMPR-sensitive nucleoside influx in guinea-pig erythrocytes than ventricular myocytes, indicating that the potency of some of the compounds studied is tissue dependent.

Adenosine↗
PubMed1994

The in situ size of the dopamine transporter is a tetramer as estimated by radiation inactivation.

Radiation inactivation analysis of the mazindol-sensitive binding of the dopamine transporter inhibitor, [3H]GBR-12935 to canine striatal membranes yielded a radiation inactivation target size of 278 +/- 16 kDa. This result, in conjunction with other findings in the literature demonstrating that the molecular mass of the dopamine transporter is approximately 70 kDa, suggests that the native form of the dopamine carrier in the neuronal membranes is a tetrameric assembly of identical 70 kDa subunits.

Animals↗
PubMed1994

Adenosine transport in cultured human umbilical vein endothelial cells is reduced in diabetes.

Adenosine transport in cultured human umbilical vein endothelial cells (HUVEC) was characterized and shown to be mediated by a single facilitated diffusion mechanism. Initial rates of adenosine influx at 22 degrees C were saturable [apparent Michaelis constant, 69 +/- 10 microM; maximum velocity (Vmax), 600 +/- 70 pmol.10(6) cells-1.s-1] and inhibited by nitrobenzylthioinosine (NBMPR). Formycin B had an unusually high affinity [inhibitory constant (Ki), 18 +/- 4.3 microM], whereas inosine had a low affinity (Ki, 440 +/- 68 microM) and nucleobases were without effect on adenosine influx. The number of transporters (1.2 x 10(6) sites/cell) was estimated by NBMPR equilibrium binding (apparent dissociation constant, 0.11 +/- 0.01 nM; maximum binding, 2.0 +/- 0.15 pmol/10(6) cells). In addition, we compared these endothelial cells with those obtained from cords from pregnancies complicated by diabetes (HUVEC-D), since embriopathy may occur in these conditions. HUVEC-D exhibited a 2.3-fold reduction in both the Vmax for adenosine influx and the maximum number of NBMPR binding sites (260 +/- 40 pmol.10(6) cells-1.s-1 and 0.86 +/- 0.08 pmol/10(6) cells, respectively). However, the turnover number for each nucleoside transporter in normal and diabetic HUVEC was similar (approximately 300 adenosine molecules/s). Adenosine metabolism at 10 microM in HUVEC-D was modified compared with normal cells. Intracellular phosphorylation (> 90%) was the predominant pathway in normal HUVEC, whereas in HUVEC-D, substantial levels of adenine and adenosine were detected. The present results demonstrate therefore the downregulation of the NBMPR-sensitive nucleoside transporter and changes in adenosine metabolism in HUVEC from diabetic pregnancies.

Adenosine↗
PubMed1993

High affinity sodium-dependent nucleobase transport in cultured renal epithelial cells (LLC-PK1).

Two distinct transporters for nucleobases have been characterized in LLC-PK1 cells. The first system accumulates hypoxanthine against a concentration gradient in the presence of sodium. The sodium-dependent uptake of hypoxanthine was saturable at 22 degrees C with a Km value of 0.79 +/- 0.43 microM, a Vmax of 15 +/- 4 pmol/mg protein/60 s, and a Na+:hypoxanthine coupling stoichiometry of 1.27 +/- 0.20. Uptake of hypoxanthine was inhibited by 5-fluorouracil, uracil, thymine, and guanine (Ki values 3-6 microM). Adenine and nucleosides were without effect. Using cell monolayers grown on a permeable filter support, Na(+)-dependent hypoxanthine uptake occurred preferentially from the apical surface. The second system exhibited no cation specificity and was saturable with a low affinity for hypoxanthine (Km of 124 +/- 22 microM) and a high Vmax of 275 +/- 38 pmol/mg protein/60 s. Adenine and guanine inhibited Na(+)-independent hypoxanthine uptake (Ki values 30 +/- 15 and 18 +/- 7 microM, respectively). Other nucleobases and nucleosides exhibited little or no inhibition of equilibrative hypoxanthine influx. Dipyridamole, dilazep, and phloridzin were effective inhibitors of Na(+)-dependent hypoxanthine uptake but had little effect on the Na(+)-independent flux. This study represents the first direct demonstration of a unique high affinity Na+ nucleobase co-transporter system in cultured animal cells.

Animals↗
PubMed1993

Na(+)-dependent and -independent uridine uptake in an established renal epithelial cell line, OK, from the opossum kidney.

The characteristics of Na(+)-dependent and Na(+)-independent uridine uptake at 22 degrees C were determined for monolayers of OK renal epithelial cells. The majority of uridine influx in subconfluent to early confluent (day 1 postconfluency) OK monolayers was mediated via a facilitated-diffusion pathway (apparent Km 160 +/- 41 microM, Vmax 610 +/- 100 pmol/mg protein per min). This system was inhibited with high affinity by nitrobenzylthioinosine (NBMPR) (IC50 value 1.5 nM) and by purine and pyrimidine nucleosides. Specific [3H]NBMPR binding sites were detected in OK monolayers (apparent Kd 0.67 +/- 0.25 nM, Bmax 90 +/- 19 fmol/mg protein) yielding a turnover number for the carrier of 112 uridine molecules/site per s at 22 degrees C. Na(+)-dependent uridine uptake was minor in subconfluent OK monolayers, but increased 8-fold with time after confluency reaching a stable plateau at 8 days postconfluency. Inhibition of Na(+)-dependent 1 microM uridine uptake by inosine, guanosine, adenosine and uridine was biphasic with approx. 40% of the total uptake inhibited with high affinity (IC50 value 2 to 14 microM). Concentrations of thymidine and cytidine up to 1 mM had no effect on Na(+)-dependent uridine uptake and no Na(+)-dependent thymidine influx by confluent OK monolayers was detected. Using cell monolayers grown on a permeable filter support, Na(+)-dependent uridine uptake occurred preferentially from the apical surface. This high affinity component of Na(+)-dependent uridine uptake is suggested to represent the Na(+)-dependent purine preferring N1 nucleoside transporter. The Na+/uridine stoichiometry for this system was consistent with 1:1. The remaining component of Na(+)-dependent uridine uptake was inhibited by some nucleosides, such as guanosine and inosine, with low affinity (IC50 values of 0.6 to 5 mM). Other nucleosides showed little specific inhibition. We propose that this component of uridine uptake represents a mutated carrier that binds nucleosides but is defective in the translocation of permeant.

Animals↗
PubMed1992

Nucleoside transport in cultured LLC-PK1 epithelia.

The transport of nucleosides by LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, was characterised. Uridine influx was saturable (apparent Km approximately 34 microM at 22 degrees C) and inhibited by greater than 95% by nitrobenzylthioinosine (NBMPR), dilazep and a variety of purine and pyrimidine nucleosides. In contrast to other cultured animal cells, the NBMPR-sensitive nucleoside transporter in LLC-PK1 cells exhibited both a high affinity for cytidine (apparent Ki approximately 65 microM for influx) and differential 'mobility' of the carrier (the kinetic parameters of equilibrium exchange of formycin B are greater than those for formycin B influx). An additional minor component of sodium-dependent uridine influx in LLC-PK1 cells became detectable when the NBMPR-sensitive nucleoside transporter was blocked by the presence of 10 microM NBMPR. This active transport system was inhibited by adenosine, inosine and guanosine but thymidine and cytidine were without effect, inhibition properties identical to the N1 sodium-dependent nucleoside carrier in bovine renal outer cortical brush-border membrane vesicles (Williams and Jarvis (1991) Biochem. J. 274, 27-33). Late proximal tubule brush-border membrane vesicles of porcine kidney were shown to have a much reduced Na(+)-dependent uridine uptake activity compared to early proximal tubule porcine brush-border membrane vesicles. These results, together with the recent suggestion of the late proximal tubular origin of LLC-PK1 cells, suggest that in vivo nucleoside transport across the late proximal tubule cell may proceed mainly via a facilitated-diffusion process.

Affinity Labels↗
PubMed1992

The volume changes associated with the operation of the 'simple' transporter.

The effects of hydrostatic pressure (0.1-50 MPa) on uridine transport mediated by the 'simple' facilitated nucleoside transporter of guinea-pig and human erythrocytes have been studied in an attempt to identify the volume changes which occur during transport. Pressure inhibited the zero-trans (influx or efflux) mode of uridine transport in guinea-pig cells significantly more (about 2.2- x) than equilibrium exchange. The equilibrium binding of 3H-nitrobenzylthioinosine, a potent specific inhibitor of nucleoside transport, to human red cells and ghosts, was not significantly altered by pressure suggesting that the permeation site was unperturbed. Thus pressure inhibited the transporter primarily by preventing the volume increase associated with the translocation step. Furthermore, the return of the 'empty' transporter was found to be rate-limiting because it required a larger increase in volume than when the transporter was loaded with substrate.

Animals↗