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 taurine in luminal membrane vesicles from rabbit proximal tubule

The uptake of taurine by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule was examined. In pars convoluta, the transport of taurine was characterized by two Na(+)-dependent (Km1 = 0.086 mM, Km2 = 5.41 mM) systems, and one Na(+)-independent (Km = 2.87 mM) system, which in the presence of an inwardly directed H(+)-gradient was able to drive the transport of taurine into these vesicles. By contrast, in luminal membrane vesicles from pars recta, the transport of taurine occurred via a dual transport system (Km1 = 0.012 mM, Km2 = 5.62 mM), which was strictly dependent on Na+. At acidic pH with or without a H(+)-gradient, the Na(+)-dependent flux of taurine was drastically reduced. In both kind of vesicles, competition experiments only showed inhibition of the Na(+)-dependent high-affinity taurine transporter in the presence of beta-alanine, whereas there was no significant inhibition with alpha-amino acids, indicating a beta-amino acid specific transport system. Addition of beta-alanine, L-alanine, L-proline and glycine, but not L-serine reduced the H(+)-dependent uptake of taurine to approx. 50%. Moreover, only the Na(+)-dependent high-affinity transport systems in both segments specifically required Cl-. Investigation of the stoichiometry indicated 1.8 Na+: 1 Cl-: 1 taurine (high affinity), 1 Na+: 1 taurine (low affinity) and 1 H+: 1 taurine in pars convoluta. In pars recta, the data showed 1.8 Na+: 1 Cl-: 1 taurine (high affinity) and 1 Na+: 1 taurine (low affinity).     

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.     

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.     

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.     

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.     

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.     

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.     

Contraluminal uptake of serine in the proximal nephron

Rabbit proximal nephron segments were microperfused in vitro to determine whether active contraluminal uptake of serine occurs in the renal proximal tubule during bath-to-lumen transport (influx) of the L- and D-isomers in the convoluted (pars convoluta) and straight (pars recta) segments. It is known that several amino acids are actively reabsorbed in the proximal nephron by a mechanism involving co-transport with sodium at the luminal membrane. There is some evidence that certain amino acids may also be accumulated across the contraluminal membrane by an energy-dependent mechanism, indicating that net reabsorption is the result of two oppositely directed active transport processes. During in vitro microperfusion of rabbit proximal nephron segments in this study, inward movement of L- and D-serine occurred in a bath-to-cell direction against a concentration gradient in the range 305-2735:1, indicating active uptake at the contraluminal membrane. The concentration gradients were maintained during influx of both isomers of serine in the proximal tubule. L-Serine accumulation by tubular cells was similar in the pars convoluta and recta, and significantly greater than that of D-serine, which was the same in both regions of the proximal tubule. The data support the conclusion that renal handling of serine involves active contraluminal uptake of the L- and D-isomers in both regions of the proximal tubule, and suggest that contraluminal events play an important role in renal handling of amino acids.     

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.     

Distribution and properties of the glycylsarcosine-transport system in rabbit renal proximal tubule. Studies with isolated brush-border-membrane vesicles.

The distribution and properties of the peptide-transport system in rabbit renal proximal tubule was examined with glycylsarcosine as the substrate and using brush-border-membrane vesicles derived from pars convoluta (outer cortex) and pars recta (outer medulla). The dipeptide was transported into these vesicles against a concentration gradient in the presence of an inward-directed H+ gradient, demonstrating the presence of a H+-coupled peptide-transport system in outer-cortical as well as outer-medullary brush-border membranes. Even though the transport was electrogenic and was energized by a H+ gradient in both membranes, the system was more active in outer medullary membranes than in outer cortical membranes. Kinetic analysis showed that, although the affinity of the transport system for glycylsarcosine was similar in both membrane preparations, the capacity of the system was significantly greater in outer medulla than in outer cortex. In addition, the pH profiles of the peptide-transport systems in these membrane preparations also showed dissimilarities. The greater dipeptide uptake in one membrane vis-à-vis the other may probably be due to the difference in the affinity of the transport system for H+ and/or the difference in peptide/H+ stoichiometry.     

Mechanism of transport of L-alanine by luminal-membrane vesicles from pars recta of rabbit proximal tubule

The characteristics of renal transport of L-alanine by luminal-membrane vesicles from proximal straight tubules (pars recta) of rabbit kidney were investigated. The following picture emerges from transport studies. Two electrogenic and Na+ requiring systems confined to this region of the nephron exist for the transport of L-alanine. In addition to Na+, the transport of L-alanine was influenced by H+. However, H+ does not substitute for Na+, but instead potentiates the Na+ effect. Modification of histidyl residues of the intact luminal-membrane vesicles by diethylpyrocarbonate (DEP), completely abolished the transient renal accumulation of L-alanine. Substrate and Na+-protection experiments suggest that histidyl residues may be at or close to the active site of the L-alanine transporter in membrane vesicles from pars recta.     

Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake.

Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.     

HCO3- transport in basolateral membrane vesicles isolated from rat renal cortex

The mechanism of HCO3- translocation across the proximal tubule basolateral membrane was investigated by testing for Na+-HCO3- cotransport using isolated membrane vesicles purified from rat renal cortex. As indicated by 22Na+ uptake, imposing an inwardly directed HCO3- concentration gradient induced the transient concentrative accumulation of intravesicular Na+. The stimulation of basolateral membrane vesicle Na+ uptake was specifically HCO3(-)-dependent as only basolateral membrane-independent Na+ uptake was stimulated by an imposed hydroxyl gradient in the absence of HCO3-. No evidence for Na+-HCO3- cotransport was detected in brush border membrane vesicles. Charging the vesicle interior positive stimulated net intravesicular Na+ accumulation in the absence of other driving forces via a HCO3(-)-dependent pathway indicating the flow of negative charge accompanies the Na+-HCO3- cotransport event. Among the anion transport inhibitors tested, 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid demonstrated the strongest inhibitor potency at 1 mM. The Na+-coupled transport inhibitor harmaline also markedly inhibited HCO3- gradient-driven Na+ influx. A role for carbonic anhydrase in the mechanism of Na+-HCO3- cotransport is suggested by the modest inhibition of HCO3- gradient driven Na+ influx caused by acetazolamide. The imposition of Cl- concentration gradients had a marked effect on HCO3- gradient-driven Na+ influx which was furosemide-sensitive and consistent with the operation of a Na+-HCO3- for Cl- exchange mechanism. The results of this study provide evidence for an electrogenic Na+-HCO3- cotransporter in basolateral but not microvillar membrane vesicles isolated from rat kidney cortex. The possible existence of an additional basolateral membrane HCO3(-)-translocating pathway mediating Na+-HCO3- for Cl- exchange is suggested.     

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.     

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.     

Translocation of fatty acids across the basolateral rat liver plasma membrane is driven by an active potential-sensitive sodium-dependent transport system

In previous studies it was shown that hepatocellular uptake of fatty acids is mediated by a specific fatty acid binding membrane protein. To determine now directly the driving forces for their entry into hepatocytes, the uptake of a representative long chain fatty acid, [3H]oleate, by basolateral rat liver plasma membrane vesicles was examined. Influx of oleate was stimulated by increasing the Na+ concentration of the medium. In the presence of an inwardly directed Na+ gradient (NaSCN, NaNO3, NaCl) oleate was accumulated during the initial uptake phase (20 s) at a concentration of 1.4-1.9-fold that at equilibrium (overshoot). This activation of influx was not observed after replacement of Na+ by Li+, K+, or choline+. Na+-dependent oleate uptake was significantly stimulated by creation of a negative intravesicular potential, either by altering the accompanying anions or by valinomycin-induced K+ diffusion potentials, suggesting an electrogenic transport mechanism. Na+-dependent fatty acid uptake was temperature dependent, with maximal overshoots occurring at 37 degrees C, and revealed saturation kinetics with a Km of 83.1 nM and Vmax of 2.9 nmol X min-1 X mg protein-1. These studies demonstrate that the carrier-mediated hepatocellular uptake of fatty acids represents an active potential-sensitive Na+-fatty acid cotransport system.     

Ba2+-sensitive K+ channels in luminal-membrane vesicles from pars convoluta of rabbit proximal tubule

This paper describes properties of 86Rb+ fluxes through a novel K+ channel in luminal-membrane vesicles isolated from pars convoluta of rabbit proximal tubule. The uptake of 86Rb+ into potassium salt loaded vesicles was specifically inhibited by Ba2+. The isotope accumulation is driven by an electrical diffusion potential as shown in experiments using these membrane vesicles loaded with anions of different membrane permeability and was as follows: gluconate greater than SO4(2-) greater than Cl-. Furthermore, the vesicles containing the channels show a cation selectivity with the order K+ greater than Rb+ greater than Li+ greater than Na+ = choline+.     

A simple and efficient method for reconstitution of amino acid and glucose transport systems from Ehrlich ascites cells

Solubilized Ehrlich cell plasma membrane proteins were incorporated into lipid vesicles in the presence of added phospholipid, using Sephadex G-50 chromatography combined with a freeze-thaw step. Liposomes formed in K+ exhibited high levels of Na+-dependent, alpha-aminoisobutyric acid uptake which was electrogenic and inhibited by other amino acids. The transport activity reconstituted was similar to that observed in native plasma membrane vesicles. In addition to transport by system A, leucine exchange activity (system L), Na+-dependent serine exchange activity (system ASC), and stereospecific glucose transport activity were also reconstituted. The latter was inhibited by D-glucose, D-galactose, cytochalasin B, and mercuric chloride. The medium used for reconstitution was critical for the recovery of Na+-dependent amino acid transport. The use of Na+ in the reconstitution procedure led to formation of liposomes which displayed little Na+-dependent and gradient-stimulated amino acid uptake. In contrast, all transport activities studied were efficiently reconstituted in K+ medium.