Transport of glutamine by rat kidney brush-border membrane vesicles.

I studied the glycosylation in vivo of a viral envelope protein, the glycoprotein of vesicular stomatitis virus (VSV), by pulse labelling of virus-infected HeLa cells with 3H-labelled monosaccharides (mannose, glucosamine). Radioactivity was incorporated into the fraction of membrane-bound polyribosomes, although metabolic conversion of [3H]-mannose into amino acids was negligible. Dissociation of bound polyribosomes revealed that the radioactively co-purified with the peptidyl-tRNA. The nascent peptides were released by alkaline hydrolysis, immunoprecipitated and analysed by polyacrylamide-gel electrophoresis. It is apparent from the size distribution of the [3H]mannose-labelled nascent chains that attachment of carbohydrate starts when approximately half of the amino acid sequence of the viral glycoprotein has been synthesized.     

L-proline transport by newborn rat kidney brush-border membrane vesicles.

The transport of L-proline was studied in brush-border membrane vesicles isolated from the kidneys of newborn rats. In contrast with the rapid initial uptake with an 'overshoot' observed in adult vesicles, uptake by the newborn vesicle was slow, showed no 'overshoot', and proline continued to accumulate at a time when the adult vesicle had already equilibrated. L-Proline transport in the newborn rat occurs by Na+-dependent and independent mechanisms. There appeared to be essentially no uptake by anti-luminal vesicles isolated from newborn rat kidney. These observations may help to explain the prolinuria that occurs in the newborn animal.     

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.     

Studies of zinc transport into brush-border membrane vesicles isolated from pig small intestine

Zinc transport into brush-border membrane vesicles was investigated by measuring uptake rates at a very short incubation time (2 seconds), during the initial linear uptake. A divalent cation chelator (EGTA) was added to the stop and washout solutions in order to remove the zinc bound to the external surface of the vesicles. Under these conditions, we showed that zinc enters the vesicles by (1) a saturable carrier-mediated process, and (2) an unsaturable pathway. The kinetic parameters we calculated were an affinity of 0.215 +/- 0.039 mM, a Jmax of 17.2 +/- 1.7 nmol.min-1.(mg protein)-1 and an unsaturable constant of 0.025 +/- 0.006 (n = 6). The imposition of an outwardly directed K+ gradient (negative inside) did not affect the Jmax value of the zinc uptake but increased the Km value significantly. This suggests that, at least a portion of zinc which crosses the membrane does not do so in a cationic form. Zinc uptake was decreased or increased according to the nature of accompanying anions (Cl-, SO4(2)-, SCN-) in the absence of any membrane potential. With highly permeant anions such as thiocyanates, zinc uptake was considerably augmented, suggesting a movement of zinc in a complexed form involving the presence of negative species. We also showed that cadmium competitively inhibited the zinc uptake; we measured a Ki value of 0.21 mM, indicating a similar affinity of cadmium for the carrier as zinc itself. By contrast, the presence of calcium had little effect on zinc entry into vesicles. The calcium ionophore A23187 had only a slight stimulating effect on zinc uptake. These results indicate that zinc and calcium transports are probably independent of each other.     

Characterization of tryptophan transport in human placental brush-border membrane vesicles.

The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity.     

Membrane-potential-dependent uptake of tryptamine by rat intestinal brush-border membrane vesicles.

The effect of membrane potential on the uptake of tryptamine, an organic cation, by rat intestinal brush-border membrane vesicles was studied. In the presence of an outwardly directed H(+)-gradient, the initial uptake of tryptamine was stimulated remarkably and the overshoot phenomenon was observed. In contrast, the uptake was depressed by an inwardly-directed H(+)-gradient. The effect of H(+)-gradient on the uptake of tryptamine was maintained in the presence of FCCP, whereas it vanished when voltage-clamped vesicles were used. Moreover, the uptake of tryptamine was linearly augmented with increase of the valinomycin-induced inside-negative K+ diffusion potential. These results suggest that tryptamine is taken up into intestinal brush-border membrane vesicles depends upon the ionic diffusion potential. The effect of several indole derivatives and amine compounds on the uptake of tryptamine was also examined. The uptake of tryptamine was inhibited by all amine compounds used, but anionic and zwitterionic compounds had no effect, suggesting that these amines interact on brush-border membrane and cause an inhibitory effect.     

Dicarboxylate transport in human placental brush-border membrane vesicles

Pathways for transport of dicarboxylic acid metabolites by human placental epithelia were investigated using apical membrane vesicles isolated by divalent cation precipitation. The presence of Na+/dicarboxylate cotransport was assessed directly by [14C]succinate tracer flux measurements and indirectly by fluorescence determinations of voltage sensitive dye responses. The imposition of an inwardly directed Na+ gradient stimulated vesicle uptake of succinate achieving levels approximately 5-fold greater than those observed at equilibrium. The increased succinate uptake was specific for Na+ as no stimulation was observed in the presence of Li+, K+ or choline+ gradients. In addition to concentrative accumulation of succinate, a direct coupling of Na+/succinate cotransport was suggested by the absence of a sizeable conductive pathway for succinate uptake and decreased succinate uptake levels associated with a more rapid decay of an imposed Na+ gradient. Na+ gradient-driven succinate uptake was not the result of parallel Na+/H+ and succinate/OH- exchange activities but was reduced by the Na+-coupled inhibitor harmaline. The voltage sensitivity of Na+ gradient-driven succinate uptake suggests Na+/succinate cotransport is electrogenic occurring with net transfer of positive charge. Substrate-specificity studies suggest the tricarboxylic acid cycle intermediates as candidates for transport by the Na+-coupled pathway. Decreasing pH increased the citrate-induced inhibition of succinate uptake suggesting divalent citrate as the preferred substrate for transport. Initial rate determinations of succinate uptake indicate succinate interacts with a single saturable site (Km 33 microM) with a maximal transport rate of 0.5 nmol/mg per min.     

Spermine uptake by rat intestinal brush-border membrane vesicles

The uptake of spermine by isolated rat intestinal brush-border membrane vesicles was studied. Uptake was biphasic, with an initial rapid uptake followed by a prolonged slower phase. Spermine uptake was not affected by a Na+ electrochemical gradient. The equilibrium uptake of spermine was considerably dependent upon the medium pH. At pH 7.5 the degree of uptake was higher than that at pH 6.5 and was inversely proportional to the extravesicular osmolarity with a relatively high binding, which was estimated by extraporation to infinite extravesicular osmolarity (zero intravesicular space), while the uptake at pH 6.5 was not altered under the various medium osmolarities. A kinetic analysis of the initial uptake rate of spermine at 37 degrees C gave a Km of 24.2 microM and Vmax of 206.1 pmol/mg protein per min. Furthermore, the uptake at 4 degrees C was nonlinear, providing evidence for saturability. These findings suggest that spermine was associated with intestinal brush-border membrane vesicles in two ways, by binding to the outside and inside of membrane vesicles. The interaction of spermine and the apical membrane can be a contributory factor in the accumulation of this polyamine in the intestine of the intact animal.     

Biotin transport in rat intestinal brush-border membrane vesicles

Transport of biotin across rat intestinal brush-border membrane was examined using the brush-border membrane vesicle (BBMV) technique. Uptake of biotin by BBMV is the result of transport of the substrate into the intravesicular space with negligible binding to membrane surfaces. In the presence of a Na+ gradient (out greater than in), transport of biotin was higher with a transient 'overshoot' phenomenon. In comparison, transport of biotin in the presence of a choline gradient (out greater than in) was lower with no 'overshoot' phenomenon. In both jejunal and ileal BBMV, the transport of biotin as a function of concentration was saturable in the presence of a Na+ gradient (out greater than in) but was linear in the presence of a choline gradient (out greater than in). Vmax of the Na+-dependent transport system was 0.88 and 0.37 pmol/mg protein per s and apparent Kt was 7.57 and 7.85 microM in jejunal and ileal BBMV, respectively. Structural analogues inhibited the transport process of biotin. Unlike the electrogenic transport of D-glucose, the transport of the anionic biotin was not affected by imposing a relatively positive intravesicular potential with the use of valinomycin and an inwardly-directed K+ gradient, suggesting that biotin transport is most probably an electroneutral process. This suggestion was further supported by studies on biotin transport in the presence of anions of different lipid permeability. The results of this study demonstrate that biotin transport across rat intestinal brush-border membrane is by a carrier-mediated, Na+-dependent and electroneutral process. Furthermore, transport of biotin is higher in the jejunum than the ileum.     

Effects of membrane potential on Na cotransports in eel intestinal brush-border membrane vesicles: Studies with a fluorescent dye

Summary The Na-dependent transport of a number of organic molecules (d-glucose,l-proline,l-alanine,l-phenylalanine) in brush-border membrane vesicles isolated from the intestine of the eel (Anguilla anguilla) was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3-diethylthiacarbocyanine iodide (DiS-C₂(5)). The experimental approach consisted of: a) generating an inside-negative membrane potential mimicking in vivo conditions: b) measuring the rate of membrane potential decay (i.e., the rate of fluorescence quenching decay) due to Na-neutral substrate cotransport. Rates of membrane potential decay showed saturation on substrate concentration andK _app values (the substrate concentration giving 50% of the maximal rate) were estimated for Na-dependent transport ofd-glucose (0,099mm),l-alanine (0.516mm),l-proline (0.118mm) andl-phenylalanine (2.04mm). The influence of an inside-negative membrane potential on the affinity of the transporter for glucose and for sodium is discussed.     

Facilitated diffusion of urate in avian brush-border membrane vesicles

Membrane transport pathways mediatingtranscellular secretion of urate across the proximal tubule wereinvestigated in brush-border membrane vesicles (BBMV) isolated fromavian kidney. An inside-positive K diffusion potential induced aconductive uptake of urate to levels exceeding equilibrium.Protonophore-induced dissipation of membrane potential significantlyreduced voltage-driven urate uptake. Conductive uptake of urate wasinhibitor sensitive, substrate specific, and a saturable function ofurate concentration. Urate uptake was trans-stimulated byurate and cis-inhibited by p-aminohippurate (PAH). Conductive uptake of PAH was cis-inhibited by urate.Urate uptake was unaffected by an outward -ketoglutarate gradient. In the absence of a membrane potential, urate uptake was similar in thepresence and absence of an imposed inside-alkaline pH gradient or anoutward Cl gradient. These observations suggest a uniporter-mediated facilitated diffusion of urate as a pathway for passive efflux acrossthe brush border membrane of urate-secreting proximal tubule cells.

     

Transport of carnosine by mouse intestinal brush-border membrane vesicles

The characteristics of carnosine (beta-alanyl-L-histidine) transport have been studied using purified brush-border membrane vesicles from mouse small intestine. Uptake curves did not exhibit any overshoot phenomena, and were similar under Na+, K+ or choline+ gradient conditions (extravesicular greater than intravesicular). However, uptake of histidine showed an overshoot phenomenon in the presence of a Na+-gradient. There was no detectable hydrolysis of carnosine during 15 min of incubation with membrane vesicles under conditions used for transport experiments. Analysis of intravesicular contents further showed the complete absence of the constituent free amino acids of carnosine, and indicates that intact carnosine is transported. Studies on the effect of concentration on peptide uptake revealed that transport occurred by a saturable process conforming to Michaelis-Menten kinetics with a Km of 9.6 +/- 1.4 mM and a Vmax of 2.9 +/- 0.2 nmol/mg protein per 0.4 min. Uptake of carnosine was inhibited by both di- and tripeptides with a maximum inhibition of 68% by glycyl-L-leucyltyrosine. These results clearly demonstrate that carnosine is transported intact by a carrier-mediated, Na+-independent process.     

Transport of carnosine by mouse intestinal brush-border membrane vesicles

The characteristics of carnosine (β-alanyl-l-histidine) transport have been studied using purified brush-border membrane vesicles from mouse small intestine. Uptake curves did not exhibit any overshoot phenomena, and were similar under Na⁺, K⁺ or choline⁺ gradient conditions (extravesicular > intravesicular). However, uptake of histidine showed an overshoot phenomenon in the presence of a Na⁺-gradient. There was no detectable hydrolysis of carnosine during 15 min of incubation with membrane vesicles under conditions used for transport experiments. Analysis of intravesicular contents further showed the complete absence of the constituent free amino acids of carnosine, and indicates that intact carnosine is transported. Studies on the effect of concentration on peptide uptake revealed that transport occurred by a saturable process conforming to Michaelis-Menten kinetics with a K_m of 9.6 ± 1.4 mM and a V_max of 2.9 ± 0.2 nmol / mg protein per 0.4 min. Uptake of carnosine was inhibited by both di- and tripeptides with a maximum inhibition of 68% by glycyl-l-leucyltyrosine. These results clearly demonstrate that carnosine is transported intact by a carrier-mediated, Na⁺-independent process.     

Disaccharide uptake by brush-border membrane vesicles lacking the corresponding hydrolases

Intestinal disaccharide uptake was studied with isolated brush-border membrane vesicles lacking the corresponding hydrolase. Either 15-day-old chick intestine, lacking both trehalase and lactase, or newborn pig intestine, lacking sucrase, was used. Both animal species yielded osmotically active vesicles capable of D-glucose/Na+ cotransport with a positive overshoot test. Vesicles from either origin gave quantitatively similar results in regard to both initial uptake rates and relative vesicle volumes. The nontransported analogs D-mannitol and L-glucose were used as diffusion markers. When tested with the appropriate disaccharidase-lacking vesicles, lactose, trehalose and sucrose exhibited uptake rates indistinguishable from those of D-mannitol and L-glucose. These uptakes were unaffected by the presence or absence of Na+, phlorizin and Tris. Chromatographic analysis confirmed the lack of hydrolysis of each disaccharide after prolonged incubation. The inescapable conclusion seems to be that intact disaccharides are not transported through the brush-border membrane, their uptake occurring through simple diffusion.     

Aboral changes in D-glucose transport by human intestinal brush-border membrane vesicles.

D-Glucose transport was investigated in isolated brush-border membrane vesicles from human small intestine. Characteristics of D-glucose transport from the jejunum were compared with that in the mid and terminal ileum. Jejunal and mid-ileal D-glucose transport was Na+-dependent and electrogenic. The transient overshoot of jejunal D-glucose transport was significantly greater than corresponding values in mid-ileum. The terminal ileum did not exhibit Na+-dependent D-glucose transport, but did exhibit Na+-dependent taurocholate transport. Na+-glucose co-transport activity as measured by tracer-exchange experiments was greatest in the jejunum, and diminished aborally. We conclude that D-glucose transport in man is Na+-dependent and electrogenic in the proximal intestine and directly related to the activity of D-glucose-Na+ transporters present in the brush-border membranes. D-Glucose transport in the terminal ileum resembles colonic transport of D-glucose.     

Proton-coupled organic cation transport in renal brush-border membrane vesicles

We had previously proposed that organic cations are transported across the brush-border membrane in the canine kidney by a H⁺ exchange (or antiport) system (Holohan, P.D. and Ross, C.R. (1981) J. Pharmacol. Exp. Ther. 216, 294–298). In the present report, we demonstrate that in brush-border membrane vesicles the transport of organic cations is chemically coupled to the countertransport of protons, by showing that the uphill or concentrative transport of a prototypic organic cation, N¹-methylnicotinamide (NMN), is chemically coupled to the flow of protons down their chemical gradient. In a reciprocal manner, the concentrative transport of protons is coupled to the counterflow of organic cations down their concentration gradient. The transport of organic cations is monitored by measuring [³H]NMN while the transport of protons is monitored by measuring changes in acridine orange absorbance. The functional significance of the coupling is that a proton gradient lowers the K_m and increases the V_max for NMN transport.     

l-Cystine transport by papain-treated rat renal brush-border membrane vesicles

In papain-treated rat renal brush-border membrane vesicles, cystine uptake was enhanced under sodium gradient conditions. This effect was not observed when sodium was equilibrated across the vesicle membrane or when sodium was completely absent from the incubation medium. The increased rate of cystine uptake occurred within the first two minutes of incubation and coincided with the period of increased flux of sodium known to occur after papain treatment. Under sodium gradient conditions, the V_max of cystine uptake by treated vesicles was 65% greater while the K_m was 25% lower than the value observed in untreated membranes. The increased cystine uptake after papain treatment occurred when medium cystine was in the electroneutral form. In the absence of a sodium gradient, cystine uptake by control membranes was insensitive to changes in membrane potential and this was unaltered after papain treatment. Exposure of the membranes to papain also resulted in a profound decrease in cystine binding which occurs in native membranes incubated with cystine. The fact that cystine uptake is unchanged under sodium equilibration and even enhanced under sodium gradient conditions suggests that the component of cystine binding is not essential for cystine transport and may represent non-specific binding to membrane proteins.     

Proton-coupled organic cation transport in renal brush-border membrane vesicles

We had previously proposed that organic cations are transported across the brush-border membrane in the canine kidney by a H+ exchange (or antiport) system (Holohan, P.D. and Ross, C.R. (1981) J. Pharmacol. Exp. Ther. 216, 294-298). In the present report, we demonstrate that in brush-border membrane vesicles the transport of organic cations is chemically coupled to the countertransport of protons, by showing that the uphill or concentrative transport of a prototypic organic cation, N1-methylnicotinamide (NMN), is chemically coupled to the flow of protons down their chemical gradient. In a reciprocal manner, the concentrative transport of protons is coupled to the counterflow of organic cations down their concentration gradient. The transport of organic cations is monitored by measuring [3H]NMN while the transport of protons is monitored by measuring changes in acridine orange absorbance. The functional significance of the coupling is that a proton gradient lowers the Km and increases the Vmax for NMN transport.