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.     

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.     

Biotin uptake mechanisms in brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex

Biotin transport was studied using brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex. An inwardly directed Na+ gradient stimulated biotin uptake into brush-border membrane vesicles and a transient accumulation of the anion against its concentration gradient was observed. In contrast, uptake of biotin by basolateral membrane vesicles was found to be Na+-gradient insensitive. Generation of a negative intravesicular potential by valinomycin-induced K+ diffusion potentials or by the presence of Na+ salts of anions of different permeabilities enhanced biotin uptake by brush-border membrane vesicles, suggesting an electrogenic mechanism. The Na+ gradient-dependent uptake of biotin into brush-border membrane vesicles was saturable with an apparent Km of 28 microM. The Na+-dependent uptake of tracer biotin was significantly inhibited by 50 microM biotin, and thioctic acid but not by 50 microM L-lactate, D-glucose, or succinate. Finally, the existence in both types of membrane vesicles of a H+/biotin- cotransport system could not be demonstrated. These results are consistent with a model for biotin reabsorption in which the Na+/biotin- cotransporter in luminal membranes provides the driving force for uphill transport of this vitamin.     

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.     

Effects of halothane on transport of 5-hydroxytryptamine by platelet membranes.

Na+-dependent uptake of 5-HT (5-hydroxytryptamine) into plasma membrane vesicles derived from bovine blood platelets and ATP-dependent 5-HT uptake into storage vesicles in platelet lysates were measured. Na+-dependent uptake was temperature-dependent, inhibited by imipramine and exhibited Michaelis-Menten kinetics (apparent Km, 0.12 +/- 0.02 microM; Vmax. 559 +/- 54 pmol/min per mg of protein. Halothane had no effect on Na+-dependent transport of 5-HT in plasma-membrane vesicles. ATP-dependent 5-HT transport into storage granules also exhibited Michaelis-Menten kinetics (apparent Km 0.34 +/- 0.03 microM; Vmax. 34.3 +/- 1.7 pmol/min per mg of protein) and was inhibited by noradrenaline (norepinephrine), but not by imipramine. Exposure of the granules to halothane resulted in a progressive decrease in Vmax. The results demonstrate a possible site for disruption of platelet function by anaesthetics.     

Na+/HCO3-co-transport in basolateral membrane vesicles isolated from rabbit renal cortex

Recent studies suggest that the major pathway for exit of HCO3- across the basolateral membrane of the proximal tubule cell is electrogenic Na+/HCO3- co-transport. We therefore evaluated the possible presence of Na+/HCO3- co-transport in basolateral membrane vesicles isolated from the rabbit renal cortex. Imposing an inward HCO3- gradient induced the transient uphill accumulation of Na+, and imposing an outward Na+ gradient caused HCO3- -dependent generation of an inside-acid pH gradient as monitored by quenching of acridine orange fluorescence, findings consistent with the presence of Na+/HCO3- co-transport. In the absence of other driving forces, generating an inside-positive membrane potential by imposing an inward K+ gradient in the presence of valinomycin caused net Na+ uptake via a HCO3- -dependent pathway, indicating that Na+/HCO3- co-transport is electrogenic and associated with a flow of negative charge. Imposing transmembrane Cl- gradients did not appreciably affect HCO3- gradient-stimulated Na+ influx, suggesting that Na+/HCO3- co-transport is not Cl- -dependent. The rate of HCO3- gradient-stimulated Na+ influx was a simple, saturable function of the Na+ concentration (Km = 9.7 mM, Vmax = 160 nmol/min/mg of protein), was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (I50 = 100 microM), but was inhibited less than 10% by up to 1 mM amiloride. We could not demonstrate a HCO3- -dependent or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive component of Na+ influx in microvillus membrane vesicles. This study thus indicates the presence of a transport system mediating electrogenic Na+/HCO3- co-transport in basolateral, but not luminal, membrane vesicles isolated from the rabbit renal cortex. Analogous to the use of renal microvillus membrane vesicles to study Na+/H+ exchange, renal basolateral membrane vesicles may be a useful model system for examining the kinetics and possible regulation of Na+/HCO3- co-transport.     

Phosphate transport by rat intestinal basolateral-membrane vesicles.

The characteristics of phosphate transport across intestinal basolateral membranes of the rat were determined by using enriched preparations in which uphill Na+-dependent D-glucose transport could not be demonstrated, but ATP-dependent Ca2+ transport was present. Phosphate transport was saturable, Na+-dependent and exhibited Michaelis-Menten kinetics. Vmax. was 51.1 +/- 4.2 pmol/10 s per mg of protein and Km was 14 +/- 3.9 microM. The transport process was electroneutral. Tracer-exchange experiments and counter-transport studies confirmed the presence of a Na+-Pi carrier at the basolateral membrane. The presence of inside-positive membrane potential did not enhance phosphate uptake, indicating that the Na+ effect is secondary to the presence of the Na+-Pi carrier rather than an induction of positive membrane potential. The stoichiometry of this carrier at pH 7.4 was 2 Na+:1 phosphate, as shown by direct studies utilizing the static-head method. These studies are the first to determine the presence of a phosphate carrier at the basolateral membrane.     

Dicarboxylate transport in renal basolateral and brush-border membrane vesicles.

Characteristics of succinate transport were determined in basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively) isolated in parallel from rabbit renal cortex. The uptake of succinate was markedly stimulated by the imposition of an inwardly directed Na+ gradient, showing an "overshoot" phenomenon in both membrane preparations. The stimulation of succinate uptake by an inwardly directed Na+ gradient was not significantly affected by pH clamp or inhibition of Na(+)-H+ exchange. The Na(+)-dependent and -independent succinate uptakes were not stimulated by an outwardly directed pH gradient. The Na dependence of succinate uptake exhibited sigmoidal kinetics, with Hill coefficients of 2.17 and 2.38 in BLMV and BBMV, respectively. The Na(+)-dependent succinate uptake by BLMV and BBMV was stimulated by a valinomycin-induced inside-negative potential. The Na(+)-dependent succinate uptake by BLMV and BBMV followed a simple Michaelis-Menten kinetics, with an apparent Km of 22.20 +/- 4.08 and 71.52 +/- 0.14 microM and a Vmax of 39.0 +/- 3.72 and 70.20 +/- 0.96 nmol/(mg.min), respectively. The substrate specificity and the inhibitor sensitivity of the succinate transport system appeared to be very similar in both membranes. These results indicate that both the renal brush-border and basolateral membranes possess the Na(+)-dependent dicarboxylate transport system with very similar properties but with different substrate affinity and transport capacity.     

Na+-dependent transport of alpha-aminoisobutyrate in isolated basolateral membrane vesicles from rat parotid glands

Basolateral plasma membranes were prepared from rat parotid gland after centrifugation in a self-orienting Percoll gradient. K+-dependent phosphatase [Na+ + K+)-ATPase), a marker enzyme for basolateral membranes, was enriched 10-fold from tissue homogenates. Using this preparation, the transport of alpha-aminoisobutyrate was studied. The uptake of alpha-aminoisobutyrate was Na+-dependent, osmotically sensitive, and temperature-dependent. In the presence of a Na+ gradient between the extra- and intravesicular solutions, vesicles showed an 'overshoot' accumulation of alpha-aminoisobutyrate. Sodium-dependent alpha-aminoisobutyrate uptake was saturable, exhibiting an apparent Km of 1.28 +/- 0.35 mM and Vmax of 780 +/- 170 pmol/min per mg protein. alpha-Aminoisobutyrate transport was inhibited considerably by monensin, but incubating with ouabain was without effect. These results suggest that basolateral membrane vesicles, which possess an active amino acid transport system (system A), can be prepared from the rat parotid gland.     

A carrier-mediated transport for folate in basolateral membrane vesicles of rat small intestine.

The mechanism of exit of folate from the enterocyte, i.e. transport across the basolateral membrane, is not known. In this study we examined, using basolateral membrane vesicles, the transport of folic acid across the basolateral membrane of rat intestine. Uptake of folic acid by these vesicles represents transport of the substrate into the intravesicular compartment and not binding to the membrane surface. The rate of folic acid transport was linear for the first 1 min of incubation but decreased thereafter, reaching equilibrium after 5 min of incubation. The transport of folic acid was: (1) saturable as a function of concentration with an apparent Km of 0.6 +/- 0.17 microM and Vmax. of 1.01 +/- 0.11 pmol/30 s per mg of protein; (2) inhibited in a competitive manner by the structural analogues 5-methyltetrahydrofolate and methotrexate (Ki = 2 and 1.4 microM, respectively); (4) electroneutral; (5) Na+-independent; (6) sensitive to the effect of the anion exchange inhibitor 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS). These data indicate the existence of a carrier-mediated transport system for folic acid in rat intestinal basolateral membrane and demonstrate that the transport process is electroneutral, Na+-independent and sensitive to the effect of anion exchange inhibition.     

Transport of glycyl-L-proline in intestinal brush-border membrane vesicles of the suckling rat: characteristics and maturation

Transport of the dipeptide glycine-L-proline (Gly-L-Pro) in the developing intestine of suckling rats and its subsequent maturation in adult rats was examined using the brush-border membrane vesicles (BBMV) technique. Uptake of Gly-L-Pro by BBMV was mainly the result of transport into the intravesicular space with little binding to membrane surfaces. Transport of Gly-L-Pro in BBMV of suckling rats was: (1) Na+ independent; (2) pH dependent with maximum uptake at an incubation buffer pH of 5.0; (3) saturable as a function of concentration (apparent Km = 21.5 +/- 7.9 mM, Vmax = 8.6 +/- 1.5 nmol/mg protein per 10 s); (4) inhibited by other di- and tripeptides; and (5) stimulated and inhibited by inducing a negative and positive intravesicular membrane electrical potential, respectively. Similarly, transport of Gly-L-Pro in intestinal BBMV of adult rats was saturable as a function of concentration (apparent Km = 17.4 +/- 8.6 mM, Vmax = 9.1 +/- 2.1 nmol/mg protein per 10 s) and was stimulated and inhibited by inducing a relatively negative and positive intravesicular membrane potential, respectively. No difference in the transport kinetic parameters of Gly-L-Pro was observed in suckling and adult rats, indicating a similar activity (and/or number) and affinity of the transport carrier in the two age groups. These results demonstrate that the transport of Gly-L-Pro is by a carrier-mediated process which is fully developed at the suckling period. Furthermore, the process is H+-dependent but not Na+-dependent, electrogenic and most probably occurs by a Gly-L-Pro/H+ cotransport mechanism.     

Pantothenate-sodium cotransport in renal brush-border membranes

The mechanism of pantothenate transport into rabbit renal brush-border membrane vesicles was studied. Under voltage-clamped conditions, an inward NaCl gradient induced the transient accumulation of pantothenate against its concentration gradient, indicating Na+/pantothenate cotransport. K+, Rb+, Li+, NH4+, and choline+ were ineffective in replacing Na+. Pantothenate analogs, D-glucose, and various carboxylic acids did not inhibit Na+-dependent pantothenate transport, suggesting that this system is specific for pantothenate. Kinetic analysis of the Na+-dependent pantothenate uptake revealed a single transport system which obeyed Michaelis-Menten kinetics (Km = 16 microM and Vmax = 6.7 pmol X mg-1 X 10 s-1). Imposition of an inside-negative membrane potential caused net uphill pantothenate accumulation in the presence of Na+ but absence of a Na+ gradient, indicating that Na+/pantothenate cotransport is electrogenic. The relationship between extravesicular Na+ concentration and pantothenate transport measured under voltage-clamped conditions was sigmoidal: a Hill coefficient (napp) of 2 and a [Na+]0.5 of 55 mM were calculated. It is suggested that an anionic pantothenate1- molecule is cotransported with two Na+ to give a net charge of +1. The coupling of pantothenate transport to the Na+ electrochemical gradient may provide an efficient mechanism for reabsorption of pantothenate in the kidney.     

Identification and reconstitution of the nucleoside transporter of CEM human leukemia cells

The major nucleoside transporter of the human T leukemia cell line CEM has been identified by photoaffinity labeling with the transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR). The photolabeled protein migrates on SDS-PAGE gels as a broad band with a mean apparent molecular weight (75,000 +/- 3000) significantly higher than that reported for the nucleoside transporter in human erythrocytes (55,000) (Young et al. (1983) J. Biol. Chem. 258, 2202-2208). However, after treatment with endoglycosidase F to remove carbohydrate, the NBMPR-binding protein in CEM cells migrates as a sharp peak with an apparent molecular weight (47,000 +/- 3000) identical to that reported for the deglycosylated protein in human erythrocytes (Kwong et al. (1986) Biochem. J. 240, 349-356). It therefore appears that the difference in the apparent molecular weight of the NBMPR-sensitive nucleoside transporter between the CEM cell line and human erythrocytes is a result of differences in glycosylation. The NBMPR-binding protein from CEM cells has been solubilized with 1% octyl glucoside and reconstituted into phospholipid vesicles by a freeze-thaw sonication technique. Optimal reconstitution of uridine transport activity was achieved using a sonication interval of 5 to 10 s and lipid to protein ratios of 60:1 or greater. Under these conditions transport activity in the reconstituted vesicles was proportional to the protein concentration and was inhibited by NBMPR. Omission of lipid or protein, or substitution of a protein extract prepared from a nucleoside transport deficient mutant of the CEM cell line resulted in vesicles with no uridine transport activity. The initial rate of uridine transport, in the vesicles prepared with CEM protein, was saturable with a Km of 103 +/- 11 microM and was inhibited by adenosine, thymidine and cytidine. The Km for uridine and the potency of the other nucleosides as inhibitors of uridine transport (adenosine greater than thymidine greater than cytidine) were similar to intact cells. Thus, although the nucleoside transporter of CEM cells has a higher molecular weight than the human erythrocyte transporter, it exhibits typical NBMPR-sensitive nucleoside transport activity both in the intact cell and when reconstituted into phospholipid vesicles.     

Intestinal calcium transport in spontaneously hypertensive rats (SHR) and their genetically matched WKY rats

Calcium transport across the basolateral membranes of the enterocyte represents the active step in calcium translocation. This step occurs by two mechanisms, an ATP-dependent pump and a Ca2+/Na+ exchange process. These studies were designed to investigate these two processes in jejunal basolateral membrane vesicles (BLMV) of the spontaneously hypertensive rats (SHR) and their genetically matched controls, Wistar-Kyoto (WKY) rats. The ATP-dependent calcium uptake was stimulated several-fold compared with no ATP condition in both SHR and WKY, but no differences were noted between rate of calcium uptake in SHR and WKY. Kinetics of ATP-dependent calcium uptake at concentrations between 0.01 and 1.0 microM revealed a Vmax of 0.67 +/- 0.03 nmol/mg protein/20 sec and a Km of 0.2 +/- 0.03 microM in SHR and Vmax of 0.69 +/- 0.12 and a Km of 0.32 +/- 0.14 microM in WKY rats. Ca2+/Na+ exchange in jejunal BLMV of SHR and WKY was investigated in two ways. First, sodium was added to the incubation medium (cis-Na+). Second, Ca2+ efflux from BLMV was studied in the presence of extravesicular Na+ (trans-Na+). Both studies suggest a decreased exchange of calcium and Na+. Kinetic parameters of Na(+)-dependent Ca2+ uptake at concentrations between 0.01 and 1.0 microM exhibited Vmax of 0.05 +/- 0.01 nanmol/mg protein/5 sec and a Km of 0.21 +/- 0.13 microM in SHR and Vmax of 0.11 +/- 0.02 nanmol/mg protein/5 sec and a Km of 0.09 +/- 0.05 in WKY, respectively. These results confirm that the intestinal BLMV of SHR and WKY rats have two mechanisms for calcium extrusion, an ATP-dependent Ca2+ transport process and a Na+/Ca2+ exchange process. The ATP-dependent process appears to be functional in SHR; however, the Ca2+/Na+ exchange mechanism appears to have a marked decrease in its maximal capacity. These findings suggest that calcium extrusion via Ca2+/Na+ is impaired in the SHR, which may lead to an increase in intracellular calcium concentration. These findings may have relevance to the development of hypertension.     

Further characterization of adenosine transport in renal brush-border membranes

Adenosine transport has been further characterized in rat renal brush-border membranes (BBM). The uptake shows two components, one sodium-independent and one sodium-dependent. Both components reflect, at least partly, translocation via a carrier mechanism, since the presence of adenosine inside the vesicles stimulates adenosine uptake in the presence as well as in the absence of sodium outside the vesicles. The sodium-dependent component is saturable (Km adenosine = 2.9 microM, Vmax = 142 pmol/min per mg protein) and is abolished at low temperatures. The sodium-independent uptake has apparently two components: one saturable (Km = 4-10 microM, Vmax = 174 pmol/min per mg protein) and one non-saturable (Vmax = 3.4 pmol/min per mg protein, Km greater than 2000 microM). Inosine, guanosine, 2-chloroadenosine and 2'-deoxyadenosine inhibit the sodium-dependent and -independent transport, as shown by trans-stimulation experiments, probably because of translocation via the respective transporter. Uridine and dipyridamole inhibited only the sodium-dependent uptake. Other analogs of adenosine showed no inhibition. The kinetic parameters of the inhibitors of the sodium-dependent component were further investigated. Inosine was the most potent inhibitor with a Ki (1.9 microM) less than the Km of adenosine. This suggests a physiological role for the BBM ecto-adenosine deaminase (enzyme which extracellularly converts adenosine to inosine), balancing the amount of nucleoside taken up as adenosine or inosine by the renal proximal tubule cell.     

The mechanism of decreased Na+-dependent D-glucose transport in brush-border membrane vesicles from rabbit kidneys with experimental Fanconi syndrome

In our previous paper (Yanase, M. et al. (1983) Biochim. Biophys. Acta 733, 95-101) we reported that the Na+-dependent D-glucose uptake into brush-border membrane vesicles is decreased in rabbits with experimental Fanconi syndrome (induced by anhydro-4-epitetracycline). In the present paper we investigate the mechanism underlying this decrease. D-Glucose is taken up into the osmotically active space in anhydro-4-epitetracycline-treated brush-border membrane vesicles and exhibits the same distribution volume and the same degree of nonspecific binding and trapping as in control brush-border membrane vesicles. The passive permeability properties of control and anhydro-4-epitetracycline-treated brush-border membrane vesicles are shown to be the same as measured by the time-dependence of L-glucose efflux from brush-border membrane vesicles. D-Glucose flux was measured by the equilibrium exchange procedure at constant external and internal Na+ concentrations and zero potential. Kinetic analyses of Na+-dependent D-glucose flux indicate that Vmax in anhydro-4-epitetracycline-treated brush-border membrane vesicles (79.3 +/- 7.6 nmol/min per mg protein) is significantly smaller than in control brush-border membrane vesicles (141.3 +/- 9.9 nmol/min per mg protein), while the Km values in the two cases are not different from each other (22.3 +/- 0.9 and 27.4 +/- 1.8 mM, respectively). These results suggest that Na+-dependent D-glucose carriers per se are affected by anhydro-4-epitetracycline, and that this disorder is an important underlying mechanism in the decreased Na+-dependent D-glucose uptake into anhydro-4-epitetracycline-treated brush-border membrane vesicles.     

Gentamicin inhibits Na+-dependent D-glucose transport in rabbit kidney brush-border membrane vesicles

We studied the effect of gentamicin on Na+-dependent D-glucose transport into brush-border membrane vesicles isolated from rabbit kidney outer cortex (early proximal tubule) and outer medulla (late proximal tubule) in vitro. We found the same osmotically active space and nonspecific binding between control and gentamicin-treated brush-border membrane vesicles. There was no difference in the passive permeability properties between control and gentamicin-treated brush-border membrane vesicles. Kinetic analyses of D-glucose transport into 1 mM gentamicin-treated brush-border membrane vesicles demonstrated that gentamicin decreased Vmax in the outer cortical preparation, while it did not affect Vmax in the outer medullary preparation. With regard to Km, there was no effect of gentamicin in any vesicle preparation. When brush-border membrane vesicles were incubated with higher concentrations of gentamicin, Na+-dependent D-glucose transport was inhibited dose-dependently in both outer cortical and outer medullary preparations. Dixon plots yield inhibition constant Ki = 4 mM in the outer cortical preparation and Ki = 7 mM in the outer medullary preparation. These results indicate that the Na+-dependent D-glucose transport system in early proximal tubule is more vulnerable to gentamicin toxicity than that in late proximal tubule.     

The role of chloride in taurine transport across the human placental brush-border membrane.

Taurine, a sulfated beta-amino acid, is conditionally essential during development. A maternal supply of taurine is necessary for normal fetal growth and neurologic development, suggesting the importance of efficient placental transfer. Uptake by the brush-border membrane (BBM) in several other tissues has been shown to be via a selective Na(+)-dependent carrier mechanism which also has a specific anion requirement. Using BBM vesicles purified from the human placenta, we have confirmed the presence of Na(+)-dependent, carrier-mediated taurine transport with an apparent Km of 4.00 +/- 0.22 microM and a Vmax of 11.72-0.36 pmol mg-1 protein 20 s-1. Anion dependence was examined under voltage-clamped conditions, in order to minimize the contribution of membrane potential to transport. Uptake was significantly reduced when anions such as thiocyanate, gluconate, or nitrate were substituted for Cl-. In addition, a Cl(-)-gradient alone (under Na(+)-equilibrated conditions) could energize uphill transport as evidenced by accelerated uptake (3.13 +/- 0.8 pmol mg-1 protein 20 s-1) and an overshoot compared to Na+, Cl- equilibrated conditions (0.60 +/- 0.06 pmol mg-1 protein 20 s-1). A Cl(-)-gradient (Na(+)-equilibrated) also stimulated uptake of [3H]taurine against its concentration gradient. Analysis of uptake in the presence of varying concentrations of external Cl- suggested that 1 Cl- ion is involved in Na+/taurine cotransport. We conclude that Na(+)-dependent taurine uptake in the placental BBM has a selective anion requirement for optimum transport. This process is electrogenic and involves a stoichiometry of 2:1:1 for Na+/Cl-/taurine symport.     

Characterization of basolateral membrane Na/H antiport in rat jejunum

Na uptake studies were performed in order to examine the activity of a Na/H exchanger in basolateral membrane vesicles isolated from rat jejunum. Experiments were carried out under voltage-clamped conditions in order to avoid electrodiffusional ionic movements. 1 mM Na uptake was found to be enhanced by an outward proton gradient and its initial rate was further increased by the presence of monensin or nigericin. The pH gradient-driven Na uptake was inhibited by 2 mM amiloride and unaffected by 0.1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The initial rate of the proton gradient-induced Na uptake was saturable with respect to external Na, with a Km of 13.6 +/- 1.4 mM and a Vmax of 35.4 +/- 2.2 nmol/mg protein per min. Li competed with Na for the exchange process, whereas K, Rb, Cs, tetramethylammonium had no effect. We conclude that rat jejunal basolateral membrane contains a Na/H exchanger whose properties are similar to those of the antiporter identified in the brush-border membrane.     

Sodium-dependent nucleoside transport in choroid plexus from rabbit. Evidence for a single transporter for purine and pyrimidine nucleosides.

The overall goal of this study was to determine the mechanisms by which nucleosides are transported in choroid plexus. Choroid plexus tissue slices obtained from rabbit brain were depleted of ATP with 2,4-dinitrophenol. Uridine and thymidine accumulated in the slices against a concentration gradient in the presence of an inwardly directed Na+ gradient. The Na(+)-driven uptake of uridine and thymidine was saturable with Km values of 18.1 +/- 2.0 and 13.0 +/- 2.3 microM and Vmax values of 5.5 +/- 0.3 and 1.0 +/- 0.2 nmol/g/s, respectively. Na(+)-driven uridine uptake was inhibited by naturally occurring ribo- and deoxyribonucleosides (adenosine, cytidine, and thymidine) but not by synthetic nucleoside analogs (dideoxyadenosine, dideoxycytidine, cytidine arabinoside, and 3'-azidothymidine). Both purine (guanosine, inosine, formycin B) and pyrimidine nucleosides (uridine and cytidine) were potent inhibitors of Na(+)-thymidine transport with IC50 values ranging between 5 and 23 microM. Formycin B competitively inhibited Na(+)-thymidine uptake and thymidine trans-stimulated formycin B uptake. These data suggest that both purine and pyrimidine nucleosides are substrates of the same system. The stoichiometric coupling ratios between Na+ and the nucleosides, guanosine, uridine, and thymidine, were 1.87 +/- 0.10, 1.99 +/- 0.35, and 2.07 +/- 0.09, respectively. The system differs from Na(+)-nucleoside co-transport systems in other tissues which are generally selective for either purine or pyrimidine nucleosides and which have stoichiometric ratios of 1. This study represents the first direct demonstration of a unique Na(+)-nucleoside co-transport system in choroid plexus.