Characteristics of D-alanine transport by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule

Uptake of D-alanine against a concentration gradient has been shown to occur with isolated luminal-membrane vesicles from pars convoluta or pars recta of rabbit proximal tubule. Renal D-alanine transport systems, displaying the following characteristics, were shown: (1) In vesicles from pars convoluta, the uptake of D-alanine was mediated by both Na+-dependent and Na+-independent transport processes. It was found that an inwardly directed H+-gradient could drive the transport of D-alanine into the vesicles both in the presence and absence of Na+. Thus, in addition to Na+, the transport of D-alanine is influenced by the H+-gradient. (2) In vesicles from pars recta, the transient accumulation of D-alanine was strictly dependent on Na+, since no 'overshoot' was ever observed in the absence of Na+. Although the Na+-dependent uptake of D-alanine was stimulated at acid pH, H+ did not substitute for Na+, as it apparently does in pars convoluta, but instead potentiated the Na+ effect. (3) Addition of L-alanine to vesicle preparations, both from pars convoluta and from pars recta, specifically inhibited renal uptake of D-alanine. A comparison between the transport characteristics of D- and L-alanine indicated that these two isomers of alanine probably share common transport systems located along the proximal tubule of rabbit kidney.     

Characteristics of D-galactose transport systems by luminal membrane vesicles from rabbit kidney

The characteristics of renal transport of D-galactose by luminal membrane vesicles from either whole cortex, pars recta or pars convoluta of rabbit proximal tubule were investigated by a spectrophotometric method using a potential-sensitive carbocyanine dye. Uptake of D-galactose by luminal membrane vesicles prepared from whole cortex was carried out by an Na+-dependent and electrogenic process. Eadie-Hofstee analysis of saturation-kinetic data suggested the presence of multiple transport systems in vesicles from whole cortex for the uptake of D-galactose. Tubular localization of the transport systems was studied by the use of vesicles derived from pars recta and pars convoluta. In pars recta, Na+-dependent transport of D-galactose and D-glucose occurred by means of a high-affinity system (half-saturation: D-galactose, 0.15 +/- 0.02 mM; D-glucose, 0.13 +/- 0.02 mM). These results indicated that the "carrier' responsible for the uptake of these hexoses does not discriminate between the steric position of the C-4 hydroxyl group of these two isomers. This is further confirmed by competition experiments, which showed that D-galactose and D-glucose are taken up by the same and equal affinity transport system by these vesicle preparations. Uptake of D-galactose and D-glucose by luminal membrane vesicles isolated from pars convoluta was mediated by a low-affinity common transport system (half-saturation: D-galactose, 15 +/- 2 mM; D-glucose, 2.5 +/- 0.5 mM). These findings strongly suggested that the "carrier' involved in the transport of monosaccharides in vesicles from pars convoluta is specific for the steric position of the C-4 hydroxyl group of these sugars and presumably interacts only with D-glucose at normal physiological concentration.     

Renal transport of neutral amino acids. Cation-dependent uptake of L-alanine by luminal-membrane vesicles.

The characteristics of L-alanine transport in luminal-membrane vesicles isolated either from whole cortex or from pars convoluta or pars recta of rabbit proximal tubules were studied by a rapid filtration technique and by a spectrophotometric method. Uptake of L-alanine by vesicles from whole cortex was mediated by both Na+-dependent and Na+-independent, but electrogenic, processes. The nature, mechanism and tubular localization of the transport systems were studied by the use of vesicles derived from pars convoluta and pars recta. In vesicles from pars recta transport of L-alanine was strictly dependent on Na+ and occurred via a dual transport system, namely a high-affinity (half-saturation 0.14 mM) and a low-affinity system (half-saturation 9.6 mM). The cation-dependent but Na+-unspecific transport system for L-alanine was exclusively localized to the pars convoluta, which also contained an Na+-preferring system of intermediate affinity (half saturation 2.1 mM). A closer examination of the mechanism of transport of L-alanine in vesicles from pars convoluta revealed that an H+ gradient (extravesicular greater than intravesicular) can drive the transport of L-alanine into the vesicles both in the presence and in the absence of Na+. The physiological importance of various L-alanine transporters is briefly discussed.     

Characteristics of uptake of short chain fatty acids by luminal membrane vesicles from rabbit kidney

The mechanisms of renal transport of short chain fatty acids by luminal membrane vesicles prepared from pars convoluta or pars recta of rabbit proximal tubule were studied by a Millipore filtration technique and by a spectrophotometric method using a potential-sensitive carbocyanine dye. Both luminal membrane vesicle preparations take up propionate and butyrate by strictly Na+-dependent transport systems, although with different characteristics. The uptake of short chain fatty acids by membrane vesicles from the pars convoluta was insensitive to changes in membrane potential, which is indicative of electroneutral transport of these compounds. Furthermore, kinetic studies showed that the Na+-dependent, but electrically silent transport of propionate is saturable (Km = 10.9 +/- 1.1 mM and Vmax = 3.6 +/- 0.2 nmol/mg protein per 20 s) and is unaffected by the presence of L- and D-lactate, indicating that these monocarboxylic acids did not share the same common transport system. In the luminal membrane vesicles from the pars recta, the uptake of propionate and butyrate was mediated by an Na+-dependent electrogenic transport process, since addition of the organic compounds to these vesicle/dye suspensions depolarized the membrane vesicles and the renal uptake of propionate and butyrate was enhanced by K+ diffusion potential induced by valinomycin. Competition experiments revealed that in contrast to the transport of propionate by vesicles from the pars convoluta, the Na+-dependent electrogenic transport of short chain fatty acids in vesicles from the pars recta occurred via the same transport system that is responsible for the reabsorption of L- and D-lactate in this region of rabbit kidney proximal tubule.     

Renal transport of monocarboxylic acids. Heterogeneity of lactate-transport systems along the proximal tubule.

The characteristics of D- and L-lactate transport in luminal-membrane vesicles derived from whole cortex, from the pars convoluta and from the pars recta of rabbit kidney proximal tubule were studied. It was found that uptake of both isomers in vesicles from whole cortex occurred by means of dual electrogenic transport systems, namely a low-affinity system and a high-affinity system. Uptake of both isomers in vesicles from the pars recta was strictly Na+-dependent and is mediated via a single high-affinity common transport system. Vesicles from the pars convoluta contained a cation-dependent but Na+-unspecific low-affinity common transport system for these compounds. The physiological importance of this system is briefly discussed.     

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.     

Mechanisms of uptake of ketone bodies by luminal-membrane vesicles

The energetics and location of renal transport of acetoacetate, beta-hydroxybutyrate, alpha-hydroxybutyrate and gamma-hydroxybutyrate by luminal-membrane vesicles from either whole cortex or pars convoluta or pars recta of rabbit proximal tubule were studied. Addition of either acetoacetate or beta-hydroxybutyrate or its analogues to dye-membrane-vesicle suspensions in the presence of Na+ gradient (extravesicular greater than intravesicular) resulted in absorbance changes indicative of depolarizing event(s). Valinomycin enhanced the Na+-dependent uptake of monocarboxylic acids, provided a K+ gradient (intravesicular greater than extravesicular) was present. By contrast, Na+-dependent uptake of these compounds was nearly abolished by ionophores that permit Na+ to pass through the luminal-membrane via another channel, either electrogenically (e.g. gramicidin D) or electroneutrally (e.g. nigericin). These results established that the Na+-dependent transport of ketone bodies and analogues by luminal-membrane vesicles is an electrogenic process. Eadie-Hofstee analysis of saturation kinetic data suggested the presence of multiple transport systems in vesicles from whole cortex for these compounds. Tubular localization of the transport systems was studied by the use of vesicles derived from pars convoluta and pars recta. In pars recta uptake of all these compounds was mediated by means of a single high affinity common transport system. Uptake of these compounds by vesicles from pars convoluta was carried out via a relatively low affinity but common transport system. The physiological importance of the transport systems is discussed.     

Na+-H+ exchange in luminal-membrane vesicles from rabbit proximal convoluted and straight tubules in response to metabolic acidosis.

Na+-H+-exchanger activity of pars convoluta and pars recta luminal-membrane vesicles prepared from the proximal tubule of acidotic and control rabbits were assayed by a rapid-filtration technique and an Acridine Orange method. Both experimental approaches revealed the existence of an antiporter, sensitive to metabolic acidosis, in pars convoluta membrane vesicles. Kinetic data, obtained with the pH-sensitive dye, showed that the Km for Na+ transport was unchanged by acidosis, whereas Vmax. for exchanger activity was increased, on an average, by 44%. The fluorescence method, in contrast with the rapid-filtration technique, was able to detect exchanger activity in pars recta membrane vesicles. The Km value for the antiporter located in pars recta is comparable with that calculated for pars convoluta membrane vesicles. By contrast, the Vmax. of this exchanger is only about 25% of that found for pars convoluta. Furthermore, metabolic acidosis apparently does not increase Na+-H+-exchanger activity of pars recta luminal-membrane vesicles.     

Na+ + Cl- -gradient-driven, high-affinity, uphill transport of taurine in human placental brush-border membrane vesicles

Uptake of taurine in human placental brush-border membrane vesicles was greatly stimulated in the presence of an inwardly-directed Na+ + Cl- -gradient and uphill transport of taurine could be demonstrated under these conditions. Na+ as well as Cl- were obligatory for this uptake and both ion gradients could energize the uphill transport. This Na+ + Cl- -gradient-dependent taurine uptake was stimulated by an inside-negative membrane potential, demonstrating the electrogenicity of the process. The uptake system was highly specific for beta-amino acids and the Km of the system for taurine was 6.5 +/- 0.4 microM.     

GTP-binding proteins in luminal and basolateral membranes from pars convoluta and pars recta of rabbit kidney proximal tubule.

The GTP-binding proteins on luminal and basolateral membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit proximal tubule have been examined. The membrane vesicles were highly purified, as ascertained by electron microscopy, by measurements of marker enzymes, and by investigating segmental-specific transport systems. The [35S]GTP gamma S binding to vesicles, and to sodium cholate-extracted proteins from vesicles, indicated that the total content of GTP-binding proteins were equally distributed on pars convoluta, pars recta luminal and basolateral membranes. The membranes were ADP-ribosylated with [32P]NAD+ in the presence of pertussis toxin and cholera toxin. Gel electrophoresis revealed, for all preparations, the presence of cholera toxin [32P]ADP-ribosylated 42 and 45 kDa G alpha s proteins, and pertussis toxin [32P]ADP-ribosylated 41 kDa G alpha i1, 40 kDa G alpha i2 and 41 kDa G alpha i3 proteins. The 2D electrophoresis indicated that Go's were not present in luminal nor in basolateral membranes of pars convoluta or pars recta of rabbit proximal tubule.     

Demonstration of Na+-selective channels in the luminal-membrane vesicles isolated from pars recta of rabbit proximal tubule

Characteristics of 22Na+ fluxes through Na+ channels in luminal-membrane vesicles isolated from either pars recta or pars convoluta of rabbit proximal tubule were studied. In NaCl-loaded vesicles from pars recta, transient accumulation of 22Na+ is observed, which is inhibited by amiloride. The isotope accumulation is driven by an electrical diffusion potential as shown in experiments using either these membrane vesicles loaded with different anions, or an outwardly directed K+ gradient with a K+ ionophore valinomycin. The vesicles containing the channel show a cation selectivity with the order Li+ greater than Na+ greater than K+. The amiloride-sensitive 22Na+ flux is dependent on intravesicular Ca2+. In NaCl-loaded vesicles from pars convoluta, no overshoot for 22Na+ uptake is observed. Furthermore, addition of amiloride to the incubation medium did not influence the uptake of 22Na+ in these vesicle preparations. It is concluded that Na+ channels are only present in pars recta of rabbit proximal tubule.     

Taurine and cell volume maintenance in the shark rectal gland: cellular fluxes and kinetics

Tissue slices of shark rectal gland are studied to examine the kinetics of the cellular fluxes of taurine, a major intracellular osmolyte in this organ. Maintenance of high steady-state cell taurine (50 mM) is achieved by a ouabain-sensitive active Na+-dependent uptake process and a relatively slow efflux. Uptake kinetics are described by two saturable taurine transport components (high-affinity, Km 60 microM; and low-affinity, Km 9 mM). [14C]Taurine uptake is enhanced by external Cl-, inhibited by beta-alanine and unaffected by inhibitors of the Na+/K+/2Cl- co-transport system. Two cellular efflux components of taurine are documented. Incubation of slices in p-chloromercuribenzene sulfonate (1 mM) reduces taurine uptake, increases efflux of taurine and induces cell swelling. Studies of efflux in isotonic media with various cation and anion substitutions demonstrate that high-K+ markedly enhances taurine efflux irrespective of cell volume changes (i.e. membrane stretching is not involved). Moreover, iso-osmotic cell swelling induced in media containing propionate is not associated with enhanced efflux of taurine from the cells. It is suggested that external K+ exerts a specific effect on the cytoplasmic membrane to increase its permeability to taurine.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

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.     

Electrolyte transport across the basolateral membrane of the parietal cells

The ion-transport properties of the basal lateral membranes of intact isolated parietal cells were studied at the cellular and subcellular level. The presence of an amiloride-sensitive Na+:H+ exchange was demonstrated in cells by proton gradient-driven Na+ uptake and by changes in cell pH as monitored by dimethylcarboxylfluorescein fluorescence both in a fluorimeter and on single isolated cells using a fluorescence microscope and an attached intensified photodiode array spectrophotometer. The presence of the Na+:H+ antiport in vesicles was shown both by intravesicular acidification monitored by acridine orange fluorescent quenching and by proton gradient-dependent Na+ uptake. The presence of Cl-:HCO-3 exchange was determined in intact cells by monitoring changes in cell pH due to Cl- uptake and was shown to be 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid- and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-sensitive. In vesicles, Cl-:HCO-3 exchange was demonstrated by Cl- flux measurement. The apparent affinities for both Cl- and HCO-3 on either side of the membrane were determined to be Km Cli = 20 mM, Km Clout = 17.5 mM, Km HCO-3in = 2.5 mM, and Km HCO-3out = 7.5 mM. A K+ conductance in cells and vesicles was demonstrated by monitoring K+ gradient-dependent 86Rb uptake. No evidence was found for the presence of a Cl- conductance in either cells or vesicles but a H+ conductance was found to be present in vesicles but not in intact cells. In the latter, by determining the effect of either Na+ or Cl- gradients on cell pH and by flux calculations it was concluded that the Cl-:HCO-3 exchange was the major passive flux mechanism for pH regulation in this cell type.     

Functional characterization of the Betaine/gamma-aminobutyric acid transporter BGT-1 expressed in Xenopus oocytes.

Betaine is an osmolyte accumulated in cells during osmotic cell shrinkage. The canine transporter mediating cellular accumulation of the osmolyte betaine and the neurotransmitter gamma-aminobutyric acid (BGT-1) was expressed in Xenopus oocytes and analyzed by two-electrode voltage clamp and tracer flux studies. Exposure of oocytes expressing BGT-1 to betaine or gamma-aminobutyric acid (GABA) depolarized the cell membrane in the current clamp mode and induced an inward current under voltage clamp conditions. At 1 mM substrate the induced currents decreased in the following order: betaine = GABA > diaminobutyric acid = beta-alanine > proline = quinidine > dimethylglycine > glycine > sarcosine. Both the Vmax and Km of GABA- and betaine-induced currents were voltage-dependent, and GABA- and betaine-induced currents and radioactive tracer uptake were strictly Na+-dependent but only partially dependent on the presence of Cl-. The apparent affinity of GABA decreased with decreasing Na+ concentrations. The Km of Na+ also depended on the GABA and Cl- concentration. A decrease of the Cl- concentration reduced the apparent affinity for Na+ and GABA, and a decrease of the Na+ concentration reduced the apparent affinity for Cl- and GABA. A comparison of 22Na+-, 36Cl--, and 14C-labeled GABA and 14C-labeled betaine fluxes and GABA- and betaine-induced currents yielded a coupling ratio of Na+/Cl-/organic substrate of 3:1:1 or 3:2:1. Based on the data, a transport model of ordered binding is proposed in which GABA binds first, Na+ second, and Cl- third. In conclusion, BGT-1 displays significant functional differences from the other members of the GABA transporter family.     

Characterization of D-Aspartic Acid Uptake by Rat Hippocampal Slices and Effect of Ischemic Conditions

The cellular uptake of D-aspartic acid (D-Asp) as a model compound for glutamic acid transport was studied in rat hippocampal slices. D-Asp is accumulated by both Na(+)-dependent and Na(+)-independent processes in hippocampal slices, and both processes are dependent on temperature. The Na(+)-dependent uptake is assumed to be high in affinity (apparent Km = 0.17 mM), but low in capacity, whereas the Na(+)-independent uptake is much lower in affinity (Km = 2.86 mM), but higher in capacity. L-Aspartic acid, L-glutamic acid, dihydrokainic acid, and threo-3-hydroxy-DL-aspartic acid markedly inhibited the uptake of D-Asp with Na+ in the medium, whereas D-glutamic acid, glycine, and L-lysine had no significant effect. The Na(+)-dependent uptake of D-Asp was significantly reduced under "hypoglycemic," "anoxic," and "ischemic" conditions, whereas the Na(+)-independent uptake was unaffected. Metabolic inhibitors such as NaCN and ICH2COOH significantly inhibited the Na(+)-dependent uptake, but not the Na(+)-independent uptake. These results suggest that the Na(+)-dependent component of D-Asp transport in rat hippocampal cells is inactivated under ischemic conditions, whereas the Na(+)-independent component is unaffected.     

Kinetics of D-glucose transport across the intestinal brush-border membrane of the cat

1. D-glucose transport across the intestinal brush-border membrane of the cat, a carnivorous animal, was investigated using isolated brush-border membrane vesicles (BBMV). Kinetic experiments were performed under zero-trans conditions (initial [Na+]in and [Gluc]in = O) with the transmembrane electrical potential difference clamped to zero. 2. D-glucose uptake by the BBMV was strongly stimulated by an inwardly directed Na+-gradient. Uptake under Na+-free conditions seemed to occur by simple diffusion. 3. The apparent kinetic constants (Vmax, Km) of Na+-dependent D-glucose transport were computed by forcing initial uptake rates at 0.002-10.0 mmol/l D-glucose to either a Michaelis-Menten type equation with a single or with two carrier-mediated components. 4. Best fit of the experimental data was obtained with the two-component model indicating the existence of two Na+-dependent carrier-mediated mechanisms. System 1 and system 2 differ with respect to the transport velocity as well as the substrate affinity constants with Vmax being 2.5-fold and Km being 5-fold higher for system 1 compared with system 2.     

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.     

Kinetic analysis of L-glutamine transport into porcine jejunal enterocyte brush-border membrane vesicles

L-Glutamine transport into porcine jejunal enterocyte brush border membrane vesicles was studied. Uptake was mediated by a Na(+)-dependent and a Na(+)-independent pathway as well as by diffusion. The initial rates of glutamine uptake over a range of concentrations is both Na(+)-gradient and Na(+)-free conditions were analyzed and kinetic parameters were obtained. Na(+)-dependent glutamine transport had a K(m) of 0.77 +/- 0.16 mM and a Jmax of 70.7 +/- 5.8 pmol mg protein-1 s-1; Na(+)-independent glutamine transport had a K(m) of 3.55 +/- 0.78 mM and a Jmax of 55.1 +/- 6.6 pmol mg protein-1 s-1. The non-saturable component measured with HgCl2-poisoned brush border membrane vesicles in the Na(+)-free condition contained passive diffusion and non-specific membrane binding and was defined to be apparent glutamine diffusion and the glutamine permeability coefficient (Kdiff) was estimated to be Kdiff = 3.78 +/- 0.06 pmol 1 mg protein-1 mmol-1 s-1. Results of inhibition experiments showed that Na(+)-dependent glutamine uptake occurred primarily through the brush border system-B degree transporters, whereas Na(+)-independent glutamine uptake occurred via the system-L transporters. Furthermore, the kinetics of L-leucine and L-cysteine inhibition of L-glutamine uptake demonstrated that neutral amino acids sharing the same brush border transporters can effectively inhibit each other in their transport.