Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles.

The sodium-dependent entry of proline and glycine into rat renal brush-border membrane vesicles was examined. The high Km system for proline shows no sodium dependence. The low Km system for glycine entry is strictly dependent on a Na+ gradient but shows no evidence of the carrier system having any affinity for Na+. The low Km system for proline and high Km system for glycine transport appear to be shared. Both systems are stimulated by a Na+ gradient and appear to have an affinity for the Na+. The effect of decreasing the Na+ concentration in the ionic gradient is to alter the Km for amino acid entry and, at low Na+ concentrations, to inhibit the V for glycine entry.     

Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles

The sodium-dependent entry of proline and glycine into rat renal brushborder membrane vesicles was examined. The high K_m system for proline shows no sodium dependence. The low K_m system for glycine entry is strictly dependent on a Na⁺ gradient but shows no evidence of the carrier system having any affinity for Na⁺. The low K_m system for proline and high K_m system for glycine transport appear to be shared. Both systems are stimulated by a Na⁺ gradient and appear to have an affinity for the Na⁺. The effect of decreasing the Na⁺ concentration in the ionic gradient is to alter the K_m for amino acid entry and, at low Na⁺ concentrations, to inhibit the V for glycine entry.     

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.     

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.     

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.     

Inhibition of alkaline phosphatase activity and D-glucose uptake in rat renal brush-border membrane vesicles by aminoglycosides

The binding of aminoglycoside antibiotics to, and their effects on, the plasma membrane were studied using isolated rat renal brush-border membrane vesicles. Dibekacin was noted to bind with brush-border membrane vesicles having a single class of many binding sites. 3H-labeled dibekacin binding was inhibited competitively by unlabeled dibekacin, gentamicin or amikacin. The inhibition constants obtained from the Dixon plots followed the order of gentamicin approximately equal to dibekacin greater than amikacin. The alkaline phosphatase activity of brush-border membrane vesicles was inhibited by gentamicin significantly, as was also observed by a histochemical study. Sodium-dependent D-glucose uptake by brush-border membrane vesicles was significantly inhibited by the addition of gentamicin.     

Binding of 125I-labelled albumin by isolated rat renal brush-border membrane vesicles. Evidence for uptake and internalization process

1. In the kidney, filtered proteins are rapidly reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with the protein binding to the luminal brush-border membrane. 2. The binding of 125I-labelled albumin to rat renal brush-border membrane vesicles and the effect of a low molecular weight protein lysozyme on that binding was assessed by the filtration method. 3. The Scatchard plot revealed a one-component binding-type curve with a dissociation constant Kd of 430.9 nM and 39.6 pmol/mg membrane protein for the number of binding sites. 4. Albumin binding was saturable and reversible, time and temperature dependent and the initial rate enhanced by increasing amounts of lysozyme. 5. The fact that association of albumin with the brush-border membrane vesicles was dependent upon the intravesicular space suggested a double process, binding of the ligand to the membrane surface and its internalization. These data suggest that albumin has a different binding site than that of a low-molecular weight protein lysozyme, with a constant affinity value near physiological loads. That specificity may confer selectivity upon the endocytic uptake process.     

Stimulation of calcium uptake by parathyroid hormone in renal brush-border membrane vesicles. Relationship to membrane phosphorylation

The effect of parathyroid hormone (PTH) on Ca2+ uptake was studied in brush-border membrane vesicles (BBMV) prepared from the kidneys of dogs administered 4-5 micrograms/kg of bovine PTH 1-84 in vivo. PTH stimulated Ca2+ uptake at 20 s of incubation from control values of 231 +/- 21 to 306 +/- 30 pmol/mg of protein, p less than 0.001. The stimulation of Ca2+ uptake by PTH was not reversed by incubation of the BBMV with the Ca2+ ionophore, despite the fact that Ca2+ uptake was several times greater than the expected uptake at equilibrium, indicating that most of the uptake represented Ca2+ binding to the BBMV. In BBMV from kidneys exposed to PTH, hypotonic lysis or increasing the osmolality of the solution external to the BBMV did not affect Ca2+ uptake. These data also indicated that the largest fraction of Ca2+ uptake in the presence of a chemical potential represented binding of Ca2+ to BBMV. Ca2+ binding was initially to the exterior of the BBMV, then translocated within the membrane and to the interior vesicular face as assessed by chelation of Ca2+ bound to the BBMV by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Incubation of BBMV from kidneys exposed to PTH with gentamicin, which competes with Ca2+ for anionic phospholipid-binding sites, reversed the stimulatory effects of PTH on Ca2+ uptake. Phosphorylation of BBMV and PTH treatment in vivo had similar effects on BBMV phospholipid composition increasing the levels of anionic phospholipids. Phosphorylation of the BBMV also produced gentamicin-inhibitable increases in membrane Ca2+ binding. Phosphorylation of BBMV from kidneys exposed to PTH was inhibited suggesting a higher state of phosphorylation in vivo. The data demonstrate that PTH administered in vivo stimulated Ca2+ binding in BBMV that was gentamicin inhibitable and associated with an increase in the membrane content of anionic phospholipids.     

Glutamine uptake by rat renal basolateral membrane vesicles

The presence of a sodium-dependent, saturable uptake process is described in basolateral membranes of rat renal cortex for L-glutamine. Concentration-dependence studies indicate the presence of multiple transport systems withK _{m 1} of 0.032 mM and V₁ of 0.028 nmol/mg of protein per min, andK _{m 2} of 17.6 mM and V₂ of 17.6 nmol/mg of protein per min. Lysine completely inhibits the high-affinity, low-capacityK _m system and partially inhibits the low-affinity, high-capacity system. Cystine and other dibasic amino acids also affect glutamine uptake.     

Na+-independent dehydro-l-ascorbic acid uptake in renal brush-border membrane vesicles

A membrane preparation enriched in the brush-border component of the plasma membrane was isolated from rat renal superficial cortex by a divalent cation precipitation procedure. Uptake of dehydro-l-ascorbic acid, the oxidized form of l-ascorbic acid, by the brush-border membrane vesicles was studied. The uptake mechanism was found to be sodium-independent and insensitive to the trans-membrane electrical potential difference. Uptake was saturable and subject to cis-inhibition. Concentrative uptake was demonstrated only under conditions of trans-stimulation by structural analogs. The results suggest a mechanism of facilitated diffusion for the uptake of dehydro-l-ascorbic acid in renal brush-border membranes.     

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.     

Sodium uptake mechanisms in brush-border membrane vesicles prepared from rabbit renal cortex

Amiloride-sensitive and amiloride-insensitive components of ²²Na⁺ uptake were examined in brush-border membrane vesicles prepared from rabbit renal cortex. Both components could be stimulated by interior-negative electrical potentials, demonstrating a sodium conductance pathway and an effect of electrical potential on the initial rate of Na⁺/H⁺ exchange.     

Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles

Thiamine, a water-soluble vitamin, is essential fornormal cellular functions, growth and development. Thiamine deficiency leads to significant clinical problems and occurs under a variety ofconditions. To date, however, little is known about the mechanism ofthiamine absorption in the native human small intestine. The objectiveof this study was, therefore, to characterize the mechanism of thiaminetransport across the brush-border membrane (BBM) of human smallintestine. With the use of purified BBM vesicles (BBMV) isolated fromthe jejunum of organ donors, thiamine uptake was found to be1) independent of Na⁺ but markedly stimulated byan outwardly directed H⁺ gradient (pH 5.5_in/pH7.5_out); 2) competitively inhibited by thecation transport inhibitor amiloride (inhibitor constant of 0.12 mM);3) sensitive to temperature and osmolarity of the incubation medium; 4) significantly inhibited by thiamine structuralanalogs (amprolium, oxythiamine, and pyrithiamine), but not byunrelated organic cations (tetraethylammonium,N-methylnicotinamide, or choline); 5) notaffected by the addition of ATP to the inside and outside of the BBMV;6) potential insensitive; and 7) saturable as afunction of thiamine concentration with an apparent Michaelis-Menten constant of 0.61 ± 0.08 µM and a maximal velocity of 1.00 ± 0.47 pmol · mg protein¹ · 10 s¹. Carrier-mediated thiamine uptake was also found inBBMV of human ileum. These data demonstrate the existence of aNa⁺-independent, pH-dependent, amiloride-sensitive,electroneutral carrier-mediated mechanism for thiamine absorption innative human small intestinal BBMV.

     

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.     

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.     

Glutamine and glutamic acid uptake by rat renal brushborder membrane vesicles

Summary Glutamine uptake by rat renal brushborder vesicles occurred via two distinct saturable processes withK _m values of 0.145 and 8.5 mM which were stimulated by both ionic and sodium gradients with a pH optimum of 6.8–7.1 Glutamic acid uptake also occurred by a two-component system withK _m values of 0.016 and 3.60 mM. Both components were stimulated specifically by a sodium gradient. The lowK _m system for glutamic acid had a pH optimum of 7.2–7.4. Glutamine entry at 0.06 mM was inhibited by a variety of amino acids at 3 mM, including dibasic amino acids, glycine, valine, and phenylalanine. Glutamic acid entry at 0.06 mM was inhibited 20–30% by 3 mM phenylalanine, valine, -aminoisobutyric acid, and glutamine. No metabolic alteration of glutamic acid was observed on incubation with membrane vesicles, but glutamine was significantly hydrolyzed to glutamic acid upon prolonged incubation. Hydrolysis of glutamine was negligible at 15 sec incubation which was employed for measurement of initial rate of entry. These studies provide support for the existence of an uptake system in the brushborder of the renal proximal tubule cell capable of handling the reabsorption of glutamine normally present in glomerular filtrate.     

Folate binding and transport by rat kidney brush-border membrane vesicles

[3H]Pteroylglutamic acid (PteGlu) uptake was studied using brush-border membrane vesicles isolated from rat kidney. Results on the uptake of [3H]PteGlu by brush-border membrane vesicles incubated in media of increasing osmolarities demonstrated that uptake was contributed by two components, intravesicular transport and membrane binding. Both the components of the uptake exhibited similar pH dependence, with maxima at pH 5.6, and were found to be saturable mechanisms with Km values of 6.7.10(-7) and 11.2.10(-7) M, respectively. These studies show that PteGlu is transported by isolated rat kidney brush-border membrane vesicles in a manner consistent with a saturable system and that a binding component may be functionally associated with this.     

Carrier-mediated transport systems of tetraethylammonium in rat renal brush-border and basolateral membrane vesicles

Transport of [3H]tetraethylammonium, an organic cation, has been studied in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. Some characteristics of carrier-mediated transport for tetraethylammonium were demonstrated in brush-border and basolateral membrane vesicles; the uptake was saturable, was stimulated by the countertransport effect, and showed discontinuity in an Arrhenius plot. In brush-border membrane vesicles, the presence of an H+ gradient ( [H+]i greater than [H+]o) induced a marked stimulation of tetraethylammonium uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was completely inhibited by HgCl2. In contrast, the uptake of tetraethylammonium by basolateral membrane vesicles was unaffected by an H+ gradient. Tetraethylammonium uptake by basolateral membrane vesicles was significantly stimulated by a valinomycin-induced inside-negative membrane potential, while no effect of membrane potential was observed in brush-border membrane vesicles. These results suggest that tetraethylammonium transport across brush-border membranes is driven by an H+ gradient via an electroneutral H+-tetraethylammonium antiport system, and that tetraethylammonium is transported across basolateral membranes via a carrier-mediated system and this process is stimulated by an inside-negative membrane potential.