Normal molecular size of the Na(+)-phosphate cotransporter and normal Na(+)-dependent binding of phosphonoformic acid in renal brush border membranes of X-linked Hyp mice

X-linked Hyp mice have a specific defect in Na(+)-dependent phosphate (Pi) transport at the renal brush border membrane (BBM). In the present study we examined the effect of the Hyp mutation on the molecular size of the Pi transporting unit and on Na(+)-dependent 14C-phosphonoformic (PFA) binding in renal BBM vesicles. By radiation inactivation analysis, we demonstrated that the molecular size of the Na(+)-Pi cotransporter is similar in normal (242 +/- 16 kDa) and Hyp mice (227 +/- 39 kDa). Moreover, while BBM Na(+)-dependent Pi transport is significantly reduced in Hyp mice (249 +/- 54 vs 465 +/- 82 pmol/mg protein/6s), genotype differences in (1) Na(+)-dependent PFA binding (1020 +/- 115 vs 1009 +/- 97 pmol/mg protein/30 min), (2) Pi-displaceable Na(+)-dependent PFA binding (605 +/- 82 vs 624 +/- 65 pmol/mg protein/6s), and (3) phosphate uptake at Na(+)-equilibrium (67 +/- 10 vs 54 +/- 7 pmol/mg protein/6s) are not apparent. The present data demonstrate that the molecular size of the renal BBM Na(+)-Pi cotransporter is normal in Hyp mice and suggest that the number of Na(+)-Pi cotransporters may not be reduced in the mutant strain.     

Energetics of the Na+-dependent transport of D-glucose in renal brush border membrane vesicles.

The energetics of the Na+-dependent transport of D-glucose into osmotically active membrane vesicles, derived from the brush borders of the rabbit renal proximal tubule, was studied by determining how alterations in the electrochemical potential of the membrane induced by anions, ionophores, and a proton conductor affect the uptake of the sugar. The imposition of a large NaCl gradient (medium is greater than vesicle) resulted in the transient uptake of D-glucose into brush border membranes against its concentration gradient. In the presence of Na+ salts of isethionate or sulfate, both relatively impermeable anions, there was no accumulation of D-glucose above the equilibrium value. With Na+ salts of two highly permeable lipophilic anions, NO3- and SCN-, the transient overshoot was enhanced relative to that with Cl-. With Na+ salts whose mode of membrane translocation is electroneutral, i.e. acetate, bicarbonate, and phosphate, no overshoot was found. These findings suggest that only anions which penetrate the brush border membrane and generate an electrochemical potential, negative on the inside, permit the uphill Na+-dependent transport of D-glucose.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Ketone body transport in renal brush border membrane vesicles

Ketone body uptake by renal brush border vesicles has been investigated. Ketone bodies enter into the brush border vesicles by a carrier-mediated process. The uptake is dependent on an Na⁺ gradient ([Na⁺]outside > [Na⁺]inside) and is electroneutral. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. A pH gradient (alkaline inside) also stimulates the ketone body uptake. Acetoacetate and 3-hydroxybutyrate share the same carrier as demonstrated by the accelerated exchange diffusion and mutual inhibitory effects.     

Peptide transport in intestinal and renal brush border membrane vesicles

A longstanding question about the possible dependence of transmembrane peptide transport on sodium has now been resolved. Recent studies with purified intestinal brush border membrane vesicles have shown that peptide transport across this membrane is Na⁺-independent and occurs by a non-concentrative mechanism. Similar studies with renal brush border membrane vesicles have established for the first time the presence of a peptide transport system in mammalian kidney. The essential characteristics of peptide transport in these two tissues are the same. However, it still remains to be seen whether a new mechanism other than the Na⁺-gradient, hitherto unrecognized, is involved in energizing the active transport of peptides in vivo in mammalian intestine and kidney.     

Cimetidine transport in rat renal brush border and basolateral membrane vesicles

The transport of cimetidine by rat renal brush border and basolateral membrane vesicles has been studied in relation to the transport system of organic cation. Cimetidine inhibited [3H]tetraethylammonium uptake by basolateral membrane vesicles in a dose dependent manner, and the degree of the inhibition was almost the same as that by unlabeled tetraethylammonium. In contrast, cimetidine inhibited the active transport of [3H]tetraethylammonium by brush border membrane vesicles more strongly than unlabeled tetraethylammonium did. In agreement with the transport mechanism of tetraethylammonium in brush border membranes, the presence of an H+ gradient ([H+]i greater than [H+]o) induced a marked stimulation of cimetidine uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was inhibited by unlabeled tetraethylammonium. These results suggest that cimetidine can share common carrier transport systems with tetraethylammonium in renal brush border and basolateral membranes, and that cimetidine transport across brush border membranes is driven by an H+ gradient via an H+-organic cation antiport system.     

Specificity of the Na+-dependent monocarboxylic acid transport pathway in rabbit renal brush border membranes

The substrate specificity of a Na+-dependent transport pathway for L-lactate was studied in rabbit renal brush border membrane vesicles. Jmax for L-lactate transport was unaffected by the presence of a fixed concentration of two different short-chain monocarboxylic acids, while the apparent Kt(Ka) for L-lactate increased, and this is compatible with competitive inhibition. The inhibitor constants ("Ki"'s) for the transport pathway for the two solutes examined closely corresponded to the respective "Ki"'s derived from a Dixon plot. A broad range of compounds were then tested as potential inhibitors of L-lactate transport, and the "Ki"'s thereby derived yielded specific information regarding optimal substrate recognition by the carrier. A single carboxyl group is an absolute requirement for recognition, and preference is given to 3 to 6 C chain molecules. Addition of ketone, hydroxyl and, particularly, amine groups at any carbon position, diminishes substrate-carrier interaction. Intramolecular forces, notably the inductive effects of halogens, may play a role in enhancing substrate-carrier interaction; however, no correlation was found between pKa and "Ki" for the substrates examined. We conclude that a separate monocarboxylic acid transport pathway, discrete from either the D-glucose, alpha or beta neutral amino-acid, or dicarboxylic acid carriers, exists in the renal brush border, and this handles a broad range of monocarboxylates.     

Perturbation of renal amino acid transport by brush border membrane vesicles in the vitamin D-deficient rat

Vitamin D deficiency is characterized by secondary hyperparathyroidism, phosphaturia, bicarbonaturia, and generalized amino aciduria. While the site at which the phosphaturia ensues has been described to occur at the apical membrane of the renal proximal tubule, no studies are available for amino aciduria. Thus, weanling rats were fed five vitamin D-deficient diets for 4-6 weeks: (i) VLC, 0.02% Ca, 0.3% P; (ii) VLC + 1,25[OH]2D, same + 500 pmole ip for 2 days; (iii) LC, 0.45% Ca, 0.3% P; (iv) HC, 2.5% Ca, 0.3% P; and (v) VLP, 1.2% cA, 0.1% P. The normal diet contained 1.2% Ca, 0.7% P, and 2.5 micrograms% vitamin D. Amino acids, serum 25[OH]D, 1,25[OH]2D, and PTH, using a specific anti-rat PTH antibody, were measured. There were 4.65 +/- 1.1- and 10 +/- 1.39-fold increases in the urinary excretion of taurine and proline, respectively, irrespective of diet. Hypocalcemia, secondary hyperparathyroidism, and increased concentrations of urinary cAMP were demonstrated in all diets, except VLP. Taurinuria and prolinuria manifested at the renal brush border membrane. There was 21-25% and 26-39% attenuation in the peak of the overshoot of Na(+)-dependent uptake of taurine and proline, respectively, that was statistically significant as compared to that of normal diets (P less than 0.01). VLC resulted in a reduction in the Vmax of taurine (VLC, 78.26 +/- 6.88 vs normal, 115.4 +/- 6.26 pmole/mg protein/min, P less than 0.01) and proline (VLC, 402.06 +/- 31.26 vs normal, 589.49 +/- 37.42 pmole/mg protein/15 sec, P less than 0.01) uptake. Acute supplementation with pharmacological doses of 1,25[OH]2D normalized the Vmax of taurine and proline uptake, without affecting their renal excretion. The VLP diet induced and increase in the Km of taurine (VLP, 58.95 +/- 1.88 microM vs normal, 39.75 +/- 2.75 microM P less than 0.01) and proline (VLP, 116.75 +/- 8.87 microM vs normal, 76.82 +/- 7.27 microM P less than 0.01) uptake, without an associated perturbation in the Vmax of uptake. We conclude that the amino aciduria of vitamin D deficiency manifests at the apical membrane of the proximal tubule by an attenuation in the Na(+)-dependent uptake of amino acids. This is associated with a reduction in the initial rate of uptake or number of active transporters in the presence of secondary hyperparathyroidism and hypocalcemia, or a decrease in the affinity of the symport in the presence of P depletion. The data suggest the interplay of multiple factors in the causation of amino aciduria.     

Parathyroid hormone inhibition of phosphate transport in renal brush border vesicles from phosphate-depleted dogs

Dietary phosphate (P_i) restriction increases renal P_i reabsorption and induces resistance to the phosphaturic action of parathyroid hormone. Na⁺-gradient-stimulated P_i transport in membrane vesicles isolated from the renal brush border of experimental animals has been shown to parallel changes in renal P_i reabsorption induced by dietary P_i restriction and in vivo administration of parathyroid hormone. Dietary P_i restriction has been shown to markedly inhibit the phosphaturic response to parathyroid hormone in rats and dogs. Parathyroid hormone has been reported not to decrease the Na⁺-gradient-stimulated transport of P_i in brush border membrane vesicles isolated from dietary P_i restricted rats unless the rats were administered an acute P_i load prior to killing, however, thyroparathyroidectomy of rats fed a low P_i diet has been reported to increase Na⁺-gradient-stimulated P_i transport. Using the dietary P_i restricted dog, we demonstrated no significant decrease in renal reabsorption of P_i in response to parathyroid hormone administration. However, significant decreases in P_i transport in brush border membrane vesicles isolated from the kidneys of dietary P_i restricted dogs were observed in response to in vivo parathyroid hormone administration. These data demonstrate that the resistance to the phosphaturic action of parathyroid hormone observed in vivo does not include resistance to the inhibitory effect of parathyroid hormone on P_i transport in brush border membrane vesicles. Thus, the data suggest that parathyroid hormone continues to alter P_i transport characteristics of the brush border membrane in states of P_i depletion despite the resistance to parathyroid hormone seen in vivo.     

Carrier-mediated transport of glycyl-L-proline in renal brush border vesicles

Renal brush border vesicles prepared from rabbit were shown to transport intact glycyl-L-proline into the intravesicular space by a Na⁺-independent, carrier-mediated process. The kinetics of hydrolysis of glycyl-L-proline by the vesicles showed that this dipeptide was extremely resistant to hydrolysis. The intravesicular concentration of glycyl-L-proline was the same in both NaCl and KCl media. Dipeptide uptake into the vesicles appeared to be via carrier-mediated transport down a concentration gradient.     

Effect of temperature and pH on phosphate transport through brush border membrane vesicles in rats

The kinetics of sodium gradient dependent phosphate uptake by the renal brush border membrane vesicles of the rat have ben studied under various conditions of temperature and pH. From 7 to 30 degrees C the Lineweaver-Burk plots are linear, and the apparent Km progressively increases from 54 to 91 microM. Above 30 degrees C, the apparent Km continues to increase to reach 135 microM at 40 degrees C, but a break is observed in the Lineweaver-Burk plots at the substrate concentration of 300 microM. The existence of this break, confirmed by the Eadie-Hofstee plot supports the hypothesis of a dual mechanism of phosphate transport, one for low concentrations of substrate with a Km of 100 microM and the other for high concentrations with a Km of approximately 240 microM. When the two components of the Eadie-Hofstee plot are analyzed according to a nonlinear regression program, these two values of Km become 70 microM and 1.18 mM, respectively. The Vmax continuously increases with temperature. However, the Arrhenius plot (In Vmax vs. 1/TK) shows an abrupt discontinuity at 23 degrees C. pH experiments were performed at 35 degrees C. In the absence of a proton gradient, increasing the pH from 6.5 to 7.5 and 8.5 decreases the apparent Km from 341 to 167 and 94 microM, respectively. When only the divalent form of phosphate is considered as the substrate, the apparent Km does not vary anymore with the pH and remains around the mean value of 105 microM. The uniformity of the apparent Km for the total phosphate uptake, when only the divalent phosphate is considered as being the substrate, suggests that this divalent form is the only one which is transported. Whatever the substrate considered, total phosphate or divalent phosphate, the highest Vmax is obtained at pH 7.5 which probably approximates the optimum pH inside the vesicles for the phosphate uptake.     

Chloroquine,a novel inhibitor of amino acid transport by rat renal brush border membrane vesicles

Summary Chloroquine is an antimalarial and antirheumatic lysosomotropic drug which inhibits taurine uptake into and increases efflux from cultured human lymphoblastoid cells. It inhibits taurine uptake by rat lung slices and affects the uptake and release of cystine from cystinotic fibroblasts. Speculations on its mode of action include a proton gradient effect, a non-specific alteration in membrane integrity, and membrane stabilization. In this study, the effect of chloroquine on the uptake of several amino acids by rat renal brush border membrane vesicles (BBMV) was examined. Chloroquine significantly inhibited the secondary active, NaCl-dependent component of 10µM taurine uptake at all concentrations tested, but did not change equilibrium values. Analysis of these data indicated that the inhibition was non-competitive. Taurine uptake was reduced at all osmolarities tested, but inhibition was greatest at the lowest osmolarity. Taurine efflux was not affected by chloroquine, nor was the NaCl-independent diffusional component of taurine transport. Chloroquine (1 mM) inhibited uptake of the imino acids L-proline and glycine, and the dibasic amino acid L-lysine. It inhibited the uptake of D-glucose, but not the neutral-amino acids L-alanine or L-methionine. Uptake of the dicarboxylic amino acids, L-glutamic acid and L-aspartic acid, was slightly enhanced. With regard to amino acid uptake by BBMV, these findings may support some of the currently proposed mechanisms of the action of chloroquine but further studies are indicated to determine why it affects the initial rate of active amino acid transport.     

Amino acid uptake by isolated renal brush border membrane vesicles in various buffers

The uptake of amino acids by isolated rat renal brush border membrane vesicles in a modified Krebs-Ringer bicarbonate buffer and a phosphate buffer was compared to the uptake in the standard membrane vesicle buffer, Tris-Hepes-mannitol. The uptake in the modified Krebs-Ringer bicarbonate buffer was similar to that in the Tris-Hepes-mannitol buffer. Removal of the ionic constituents other than NaCl and NaHCO3 in the modified Krebs-Ringer bicarbonate buffer (KCl, CaCl2, KH2PO4 and MgSO4) did not affect the amino acid uptake by the isolated membrane vesicles. The timed uptake of proline under sodium gradient conditions in a phosphate buffer had a markedly dampened overshoot. Kinetic analysis of the initial rate of proline uptake in a phosphate buffer compared to a Tris-Herpes-mannitol buffer showed two entry systems for proline in each buffer with similar Km values, but the maximal rate of transport (V) for each system in the phosphate buffer was much lower than that in the Tris-Hepes-mannitol buffer. From these data, phosphate buffer does not appear to be a suitable medium for the study of amino acid uptake by isolated brush border membrane vesicles.     

Proximo-distal gradient of Na+-dependent D-glucose transport activity in the brush border membrane vesicles from the human fetal small intestine

Brush-border membrane vesicles were isolated from the jejunum and ileum of 17-20-week-old normal human fetuses and found to be highly enriched in sucrase activity with less than 5% contamination by basolateral membranes. Time course studies of D-glucose uptake clearly showed a transient, phlorizin-sensitive, and Na+-dependent accumulation of sugar into these vesicles. Higher maximum overshoot values and initial rates of D-glucose uptake were recorded in jejunal as compared to ileal vesicles while low substrate binding to the membranes, identical intravesicular volumes and equivalent dissipation of the Na+-gradient were found in the two preparations. It was concluded that a fully functional Na+-D-glucose cotransport system is present with a proximo-distal gradient of activity during the early gestation period.     

Transport ofl-cysteine by rat renal brush border membrane vesicles

Brush border membranes were isolated from rat renal cortex by a divalent cation precipitation method. L-35S-cysteine uptake into the vesicles was measured by a rapid filtration method. Only minimal binding of the amino acid to the vesicles was observed. Sodium stimulates L-cysteine uptake specifically. Anion replacement experiments, experiments in the presence of potassium/valinomycin-induced diffusion potential as well as experiments with a potential-sensitive fluorescent dye document an electrogenic sodium-dependent uptake mechanism for L-cysteine. Tracer replacement experiments as well as the fluorescence experiments indicate a preferential transport of L-cysteine. Transport of L-cysteine is inhibited by L-alanine and L-phenylalanine but not by L-glutamic acid and the L-basic amino acids. Initial, linear influx kinetics provide evidence for the existence of two transport sites. The results suggest (a) sodium-dependent mechanism(s) for L-cysteine shared by other neutral amino acids.     

Temperature dependence of solute transport and enzyme activities in hog renal brush border membrane vesicles

The temperature dependence of sodium-dependent and sodium-independent d-glucose and phosphate uptake by renal brush border membrane vesicles has been studied under tracer exchange conditions. For sodium-dependent d-glucose and phosphate uptake, discontinuities in the Arrhenius plot were observed. The apparent activation energy for both processes increased at least 4-fold with decreasing temperature. The most striking change in the slope of the Arrhenius plot occurred between 12 and 15°C. The sodium-independent uptake of d-glucose and phosphate showed a linear Arrhenius plot over the temperature range tested (35–5°C). The behavior of the transport processes was compared to the temperature dependence of typical brush border membrane enzymes. Alkaline phosphatase as intrinsic membrane protein showed a nonlinear Arrhenius plot with a transition temperature at 12.4°C. Aminopeptidase M, an extrinsic membrane protein exhibited a linear Arrhenius plot. These data indicate that the sodium-glucose and sodium-phosphate cotransport systems are intrinsic brush border membrane proteins, and that a change in membrane organization alters the activity of a variety of intrinsic membrane proteins simultaneously.