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.     

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.

     

Effect of metabolic acidosis on phosphate transport by the renal brush-border membrane

Metabolic acidosis produces a phosphaturia which is independent of parathyroid hormone or dietary phosphorus intake. To study the underlying mechanism, inorganic phosphate (P_i) and glucose transport were studied in brush-border membrane vesicles prepared from the renal cortex of parathyroidectomized rats gavaged for three days with either 7.5 ml of 1.6% NaCl (control) or 1.5% NH₄Cl (acidosis). At killing, blood pH and plasma bicarbonate were $\text{7.36 ± 0.01}$ and $\text{21.8 ± 0.8}\text{mequiv./l}$, respectively, in control and $\text{7.12 ± 0.03}$ ($\text{P < 0.01}$) and $\text{11.1 ± 1.2}$ ($\text{P < 0.01}$) in acidotic rats. Serum P_i was similar in both groups, while 24 h urine P_i excretion was higher in the acidotic group ($\text{P < 0.01}$). Peak sodium-dependent uptake of P_i, measured after 1.5 min of incubation, was higher in controls than acidotic rats ($\text{4442 ± 464}$ vs. $\text{2412 ± 259}\text{pmol/mg}$ protein, $\text{P < 0.01}$), whereas peak glucose uptake at 1.5 min was not significantly different between the groups. Equilibrium values for P_i and glucose uptake were similar in the two groups. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for P_i uptake in the control and acidotic animals were not different, 0.036 and 0.040 mM, respectively. By contrast, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ was higher in controls than in the acidotic group, 3.13 vs. 1.15 nmol/mg protein per 15 s. These results suggest that metabolic acidosis directly inhibits P_i uptake by the brush border of the proximal tubule by decreasing the availability of P_i carriers of the renal brush-border membrane.     

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.     

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.     

Role of N-acetylneuraminic acid in rat renal brush-border membrane vesicle aggregation by aminoglycosides

Interaction between aminoglycosides (AGs) and rat renal brush-border membrane (BBM) vesicles was investigated by the aggregation technique. The order of aggregation was gentamicin greater than dibekacin not equal to netilmicin greater than amikacin, and this order corresponds to the strength of the nephrotoxicity of the aminoglycosides in vivo rather than the number of amino groups in the aminoglycosides. BBM vesicles were aggregated through ionic interaction, as evident from the finding that aggregation ceased to occur at alkaline pH. By addition of N-acetylneuraminic acid (NANA) to the incubation medium, the vesicle aggregation induced by gentamicin was significantly inhibited. To affect the liberation of the NANA residue from BBM vesicles, the vesicles were treated with neuraminidase, resulting in an about 60% release with a significant decrease in the uptake of gentamicin into the vesicles. The decrease in the degree of vesicle aggregation was in proportion to the amount of NANA liberated. It follows from the findings that the NANA residue may in some way be responsible for the accumulation of aminoglycosides in renal proximal tubular cells.     

Radiation inactivation studies of the renal brush-border membrane phlorizin-binding protein

Renal brush-border membrane vesicles were irradiated in the frozen state with a high energy electron beam. The integral membrane proteins, alkaline phosphatase and 5'-nucleotidase, each showed a single exponential loss of activity with radiation dose, indicating target sizes of 67,000 and 58,000 daltons, respectively. Inactivation of sodium-dependent phlorizin binding to the brush-border membrane D-glucose transporter was more complex. One-half of the phlorizin binding sites were lost after even the smallest doses of radiation suggestive of large functional units (greater than 4 X 10(6) daltons) for a subpopulation of phlorizin binding proteins. The remaining sites behaved as a single radiation target of 110,000 +/- 8,000 daltons and retained the kinetic characteristics commonly associated with phlorizin binding to the glucose transporter. Thus, the data are consistent with the assignment of a molecular weight of 110,000 to the phlorizin binding moiety of the brush-border membrane D-glucose transport protein.     

Proton-coupled transport of glycylglycine in rabbit renal brush-border membrane vesicles

Transport of glycylglycine into rabbit renal brush-border membrane vesicles was found to be Na+-independent, H+ gradient-dependent and electrogenic. Marked overshoot uptake of the dipeptide was observed when an inward-directed proton gradient and inside-negative potential difference were imposed simultaneously across the vesicular membranes. Saturable depolarization of vesicular membranes could be demonstrated with glycylglycine by use of a fluorescent cyanine dye, di-S-C3(5). The results indicate that glycylglycine is contransported with H+ across the membranes.     

The Na(+)-H+ exchanger of the placental brush-border membrane is pharmacologically distinct from that of the renal brush-border membrane

We have compared the pharmacological properties of the human placental brush-border membrane Na(+)-H+ exchanger with those of the rabbit renal brush-border membrane Na(+)-H+ exchanger. The exchanger activity in both preparations was inhibited by cimetidine, clonidine, and harmaline. Cimetidine was found to be 4-5 times more potent than clonidine in inhibiting the placental Na+-H+ exchanger. However, the order of potency was reversed for the renal exchanger, in which case clonidine was 3-4 times more potent than cimetidine as an inhibitor. There was, however, no difference between the potencies of harmaline to inhibit the two exchangers. When amiloride and four of its analogs were tested as inhibitors, the Na(+)-H+ exchanger of the placental brush-border membrane exhibited greater sensitivity to inhibition by all of these compounds than the Na(+)-H+ exchanger of the renal brush-border membrane. The difference between the two exchangers was more prominent with the 5-amino-substituted amiloride derivatives than with amiloride. The greatest difference between the Ki values was for dimethylamiloride (the kidney/placenta ratio was 185), followed by ethylisopropyl amiloride, hexamethylene amiloride, and t-butyl amiloride. These results indicate that the two Na+-H+ exchangers are pharmacologically distinct.     

Stimulation of the efflux of L-glutamate from renal brush-border membrane vesicles by extravesicular potassium

The rate of efflux of L-glutamate from renal brush-border membrane vesicles was enhanced by Na+ and by extravesicular L-glutamate, but not by D-glutamate nor analogs of L-glutamate that do not share the Na+-L-glutamate co-transport system. These results suggest that efflux was mediated by the Na+-L-glutamate carrier. The efflux of L-glutamate was increased by extravesicular K+ or Rb+ but not by Li+, choline+, or Tris+. These findings, together with previous results showing that intravesicular K+ or Rb+ increased L-glutamate uptake and that a K+ gradient energized the concentrative uptake of the acidic amino acid in the absence of other gradients, provide evidence consistent with the hypothesis that the co-transport of Na+-L-glutamate is coupled to the transmembrane flux of K+.     

Direct evidence for the role of the membrane potential in glutathione transport by renal brush-border membranes

Transport of GSH was studied in isolated rat kidney cortical brush-border membrane vesicles in which gamma-glutamyltransferase had been inactivated by a specific affinity labeling reagent, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Transport of intact 2-3H-glycine-labeled GSH occurred into an osmotically active intravesicular space of AT-125-treated membranes. The initial rate of transport followed saturation kinetics with respect to GSH concentrations; an apparent Km of 0.21 mM and Vmax of 0.23 nmol/mg protein X 20 were calculated at 25 degrees C with a 0.1 M NaCl gradient (vesicle inside less than vesicle outside). Sodium chloride in the transport medium could be replaced with KCl without affecting transport activity. The rate of GSH uptake was enhanced by replacing KCl in the transport medium with K2SO4, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. The rate of GSH transport markedly decreased in the absence of a K+ gradient across the vesicular membranes and was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). These results indicate that GSH transport is dependent on membrane potential and involves the transfer of negative charge. The rate of GSH transport was inhibited by S-benzyl glutathione but not by glycine, glutamic acid, and gamma-glutamyl-p-nitroanilide. When incubated with [2-3H]glycine-labeled GSH, intact untreated vesicles also accumulated radioactivity; the rate of uptake was significantly higher in a Na+ gradient than in a K+ gradient. Sodium-dependent transport, but not sodium-independent uptake, was almost completely inhibited by a high concentration of unlabeled glycine. At equilibrium, most of the radioactivity which accumulated in the intravesicular space was accounted for by free glycine. These results suggest that GSH which is secreted into the tubular lumen by a specific translocase in the lumenal membranes or filtered by the glomerulus may be degraded in situ by membranous gamma-glutamyltransferase and peptidase activities which hydrolyze peptide bonds of cysteinylglycine and its derivatives. The resulting free amino acids can be reabsorbed into tubule cells by sodium-dependent transport systems in renal cortical brush-border membranes.     

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.     

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.     

Proton gradient-dependent transport of glycine in rabbit renal brush-border membrane vesicles

This study describes evidence for the existence of a H+/glycine symport system in rabbit renal brush-border membrane vesicles. An inward proton gradient stimulates glycine transport across the brush-border membrane, and this H+-driven glycine uptake is attenuated by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It is a positive rheogenic process, i.e. the H+-dependent glycine uptake is further enhanced by an intravesicular negative potential. Glycine uptake is stimulated to a lesser degree by an inward Na+ gradient. H+-dependent glycine uptake is inhibited by sarcosine (69%), an analog amino acid, imino acids (proline 81%, hydroxy proline 67%), and beta-alanine (31%), but not by neutral (L-leucine) or basic (L-lysine) amino acids. The results demonstrate that H+ glycine co-transport system in rabbit renal brush-border membrane vesicles is a carrier-mediated electrogenic process and that transport is shared by imino acids and partially by beta-alanine.     

Sulfate transport by mouse renal cortical slices does not represent uptake by brush-border membrane

We measured uptake of isotopically ³⁵S-labelled sulfate anion by slices and by brush-border membrane vesicles prepared from mouse renal cortex to identify: (i) whether metabolic incorporation of anion influences net transport; (ii) which membrane is primarily exposed in the renal cortex slice. Slices accumulated sulfate without significant incorporatoin into metabolic pools. Net uptake of sulfate at 0.1 mM by the slice occurred against an electrochemical gradient as determined by mesurement of free intracellular sulfate concentration, the isotopic distribution ratio at steady-state, and the distribution of lipophilic ions (TPP⁺ and SCN^?). Carrier mediation of sulfate transport in the slice was confirmed by observing concentration-dependent saturation of net uptake and counter-transport stimulation of efflux. Anion uptake was Na⁺-independent, K⁺- and H⁺-stimulated, and inhibited by disulfonated stilbenes. Brush-border membrane vesicles accumulated sulfate by a saturable mechanism dependent on a Na⁺ gradient (outside > inside); others have shown that uptake of sulfate by brush-border membrane vesicles is insensitive to inhibition by disulfonated stilbenes. These findings indicate that different mechanisms serve sulfate transport in renal cortex slice and brush-border membrane vesicle preparations. They also imply that the slice exposes an epithelial surface different from the brush-border, presumably the basolateral membrane, or its equivalent, since sulfate transport by slices resembles that obserbed with isolated basolateral membrane vesicles.     

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.     

Ion dependence of cystine and lysine uptake by rat renal brush-border membrane vesicles.

The shared transport system for uptake of L-cystine and L-lysine was examined in isolated rat renal brush-border membrane vesicles for the ionic requirements for activation of the system. No requirement for sodium was seen for either cystine or lysine influx. However, the efflux of lysine from the vesicle was stimulated by Na+. Therefore, the transport system appears to be asymmetric in its requirement for sodium. Two different divalent cations were used in the membrane isolations which resulted in different responses of cystine uptake to the electrogenic movement of K+ out of the vesicle. Membranes prepared by Mg-aggregation showed no stimulation of cystine influx by the imposition of a transient interior negative potential while vesicles prepared by Ca-aggregation did respond to electrogenic stimulation by an outwardly directed K-diffusion potential in the presence of valinomycin. Lysine influx was stimulated by electrogenic potassium efflux in both Mg-prepared and Ca-prepared membranes. No difference in sodium requirement for cystine influx was seen between the vesicles isolated by different cation-aggregation methods.