Effect of dietary phosphate intake on phosphate transport by isolated rat renal brush-border vesicles

Renal brush-border membrane vesicles isolated from rats kept for 6-8 weeks on a low-phosphate diet (0.15% of dry matter) showed a markedly faster Na(+)-dependent phosphate uptake than did membrane vesicles isolated from animals kept on a high-phosphate diet (2% of dry matter). Phosphate-uptake rate by brush-border membrane vesicles isolated from animals on a low-phosphate diet remained significantly increased after acute parathyroidectomy. Dietary adaptation was also observed in animals that had been parathyroidectomized before exposure to the different diets. In animals on the low-phosphate diet parathyrin administration inhibited phosphate uptake by brush-border vesicles only if the animals were repleted with P(i) (5ml of 20mm-NaH(2)PO(4)) 1h before being killed. After acute phosphate loading and parathyrin administration the difference in the transport rate between the two dietary groups remained statistically significant. The results suggest that the adaptation of proximal-tubule phosphate transport to dietary intake of phosphate is reflected in the Na(+)/phosphate co-transport system located in the luminal membrane of the proximal-tubule cell. Since the dietary effects on phosphate transport by brush-border membranes are only partially reversed by acute changes in parathyrin concentration and are also observed in chronically parathyroidectomized animals, the adaptation of the Na(+)/phosphate co-transport system to dietary phosphate intake seems to involve an additional mechanism independent of parathyrin.     

Phosphate transport by rat intestinal basolateral-membrane vesicles.

The characteristics of phosphate transport across intestinal basolateral membranes of the rat were determined by using enriched preparations in which uphill Na+-dependent D-glucose transport could not be demonstrated, but ATP-dependent Ca2+ transport was present. Phosphate transport was saturable, Na+-dependent and exhibited Michaelis-Menten kinetics. Vmax. was 51.1 +/- 4.2 pmol/10 s per mg of protein and Km was 14 +/- 3.9 microM. The transport process was electroneutral. Tracer-exchange experiments and counter-transport studies confirmed the presence of a Na+-Pi carrier at the basolateral membrane. The presence of inside-positive membrane potential did not enhance phosphate uptake, indicating that the Na+ effect is secondary to the presence of the Na+-Pi carrier rather than an induction of positive membrane potential. The stoichiometry of this carrier at pH 7.4 was 2 Na+:1 phosphate, as shown by direct studies utilizing the static-head method. These studies are the first to determine the presence of a phosphate carrier at the basolateral membrane.     

Effect of parathyrin on the transport properties of isolated renal brush-border vesicles.

The transport properties of brush-border membrane vesicles isolated by a calcium-precipitation method from the renal cortex of normal and parathyrin (parathyroid hormone)-treated rats were studied by a rapid-filtration technique. Parathyrin elicited a dose-dependent decrease in the Na+-dependent phosphate uptake by the brush-border membrane vesicles, but the uptake of D-glucose, Na+ and mannitol was not affected. A maximum inhibition of 30% was observed after the application of 30 U.S.P. units intramuscularly 1 h before the animals were killed. Intravenous infusion of dibutyryl cyclic AMP (0.5-1.5 MG) also decreased the phosphate uptake by the brush-border vesicles. Both dibutyryl cyclic AMP and parathyrin were ineffective when added in vitro to brush-border membrane vesicles isolated from normal rats. These data suggest that parathyrin exerts its action on the phosphate reabsorption in the renal proximal tubule by affecting the Na+/phosphate co-transport system in the brush-border membrane. The effects of parathyrin on Na+ and glucose transport, however, seem to be due to alterations to the driving forces for transport and not to the brush-border transport systems.     

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.     

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.     

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.     

Phosphate transport into brush-border membrane vesicles isolated from rat small intestine.

Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.     

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.     

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.     

In vitro ethanol effects on the transport properties of isolated renal brush-border membrane vesicles

Summary Thein vitro effect of ethanol on membrane structure and transport properties was studied in isolated renal brush border membrane vesicles.³¹P-NMR studies showed a dose-dependent increase in the quantity of an isotropic, possibly inverted-micellar component of the renal brush-border membrane as a result of treatment with ethanol. Such structures have been shown to be instrumental in the translocation of material across membrane bilayers. A²³Na-NMR study of Na⁺ exchange in artificial phosphatidylcholine liposomes indicated that ethanol (0.1%) was capable of rending the otherwise inert vesicles permeable to sodium, supporting the idea that ethanol may exert its action via a direct effect on the structure of the phospholipid bilayer. In the isolated renal brush-border membrane vesicles, like in the artificial liposomes, amiloride-insensitive pathways of Na⁺ transport were shown to be markedly activated by ethanol. These results were consistent with the inhibitory effect ethanol had on Na⁺ gradient-dependent transport systems such as the Na⁺ gradient-dependentd-glucose transport and Na⁺/H⁺ exchange. In conclusion, our results indicate that ethanol exerts its effect on the renal brush-border membrane by causing a structural change in the phospholipid bilayer which activates sodium intake. The inhibitory effect of ethanol on glucose uptake and Na⁺/H⁺ exchange is secondary, as a result of the dissipation of the energy-producing Na⁺ gradient.     

Glycyl-L-proline transport in rabbit enterocyte basolateral-membrane vesicles.

The properties of a peptide-transport system in rabbit enterocyte basolateral membrane were examined with glycyl-L-proline as the substrate. Basolateral-membrane vesicles prepared from rabbit proximal intestine were characterized in terms of both purity and orientation. Marker-enzyme assays show that the basolateral-membrane marker, ouabain-sensitive K(+)-activated phosphatase, is enriched 17-fold with respect to the initial homogenate. The activities of enzymes used as markers for other membranes and organelles are low, and contamination of the final membrane fraction with these is minimal. The use of immunoblotting techniques further confirms the absence of brush-border-membrane contamination. Proteins in the basolateral-membrane vesicle preparation gave no cross-reaction with antibodies against the 140 kDa antigen and the Na+/glucose-symport protein, markers specific to the brush-border membrane of the enterocyte. Conversely, antibodies raised against the classical basolateral-membrane marker, the RLA class I histocompatibility complex, reacted strongly with a 43 kDa basolateral-membrane protein. The orientation of the basolateral-membrane vesicles was shown to be predominantly inside-out on determination by two independent criteria. The uptake of [1-14C]glycyl-L-proline by these vesicles is stimulated by the presence of an inwardly directed pH gradient, and this stimulation can be abolished by the proton ionophores carbonyl cyanide p-trichloromethoxyphenylhydrazone (CCCP) and tetrachlorotrifluoromethylbenzimidazole (TTFB). Transport is also inhibited by HgCl2, thimerosal, Na+ and other glycyl dipeptides.     

The effect of azaserine on glutamine uptake by rat renal brush-border membranes.

Azaserine added directly to isolated rat renal brush-border membrane vesicles inhibits uptake of L-glutamine. Azaserine acts as a non-competitive inhibitor of the low-Km system for glutamine transport, but has no effect on the high-Km system. Preincubation of the vesicles with azaserine at 37 degrees C min is not required for transport inhibition to occur, although it is a requirement for gamma-glutamyl transpeptidase inhibition. Removal of azaserine from the vesicle preparation by repeated resuspensions in buffer results in a reversal of the transport inhibition but not in reversal of enzyme inhibition. Azaserine also inhibits vesicle uptake of L-proline and alpha-methyl D-glucoside, indicating a generalized effect on membrane transport systems. The data cast doubt on the postulate that gamma-glutamyl transpeptidase might act as the carrier mechanism for glutamine reabsorption by renal cortical cells.     

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.     

Carrier-mediated transport of cephalexin via the dipeptide transport system in rat renal brush-border membrane vesicles

Carrier-mediated transport of aminocephalosporin antibiotics by renal brush-border membrane vesicles has been studied in relation to the transport systems for dipeptides and amino acids. Dipeptides such as L-carnosine (beta-alanyl-L-histidine) and L-phenylalanylglycine competitively inhibited the uptake of cephalexin, but amino acids did not. Cephalexin uptake was stimulated by the countertransport effect of L-carnosine in the normal and papain-treated vesicles, and by the effect of L-phenylalanylglycine only in the papain-treated vesicles. In the papain-treated vesicles, the hydrolysis of dipeptides was markedly decreased, and the specific activity for cephalexin transport was increased approx. 2-fold because of the partial removal of membrane proteins. These results suggest that carrier-mediated transport of cephalexin can be transported by the system for dipeptides in renal brush-border membranes.     

Ontogeny of glutamine transport by rat liver plasma membrane vesicles.

Glutamine metabolism in the liver is essential for gluconeogenesis and ureagenesis. During the suckling period there is high hepatic protein accretion and the portal vein glutamine concentration is twice that in the adult, whereas hepatic vein glutamine concentration is similar between adult and suckling rats. Therefore, we hypothesized that glutamine uptake by the liver could be greater in the suckling period compared to the adult period. The present studies were, therefore, designed to investigate the transport of glutamine by plasma membranes of rat liver during maturation (suckling--2-week old, weanling--3-week old and adult--12-week old). Glutamine uptake by the plasma membranes of the liver represented transport into an osmotically sensitive space in all age groups. Inwardly directed Na+ gradient resulted in an "overshoot" phenomenon compared to K+ gradient. The magnitude of the overshoot was greater in suckling rats plasma membranes compared to adult membranes. Glutamine uptake under Na+ gradient was electrogenic and maximal at pH 7.5, whereas uptake under K+ gradient was electroneutral. Glutamine uptake with various concentrations of glutamine under Na+ gradient was saturable in all age groups with a Vmax of 1.5 +/- 0.1, 0.7 +/- 0.1 and 0.5 +/- 0.06 nmoles/mg protein/10 seconds in suckling, weanling and adult rats, respectively (P < 0.01). Km values were 0.6 +/- 0.1, 0.5 +/- 0.1 and 0.5 +/- 0.1 mM respectively. Vmax for Na(+)-independent glutamine uptake were 0.6 +/- 0.1, 0.55 +/- 0.07 and 0.54 +/- 0.06 nmoles/mg protein with Km values of 0.54 +/- 0.2, 0. +/- 0.1 and 0.5 +/- 0.2 mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biotin transport in rat intestinal brush-border membrane vesicles

Transport of biotin across rat intestinal brush-border membrane was examined using the brush-border membrane vesicle (BBMV) technique. Uptake of biotin by BBMV is the result of transport of the substrate into the intravesicular space with negligible binding to membrane surfaces. In the presence of a Na+ gradient (out greater than in), transport of biotin was higher with a transient 'overshoot' phenomenon. In comparison, transport of biotin in the presence of a choline gradient (out greater than in) was lower with no 'overshoot' phenomenon. In both jejunal and ileal BBMV, the transport of biotin as a function of concentration was saturable in the presence of a Na+ gradient (out greater than in) but was linear in the presence of a choline gradient (out greater than in). Vmax of the Na+-dependent transport system was 0.88 and 0.37 pmol/mg protein per s and apparent Kt was 7.57 and 7.85 microM in jejunal and ileal BBMV, respectively. Structural analogues inhibited the transport process of biotin. Unlike the electrogenic transport of D-glucose, the transport of the anionic biotin was not affected by imposing a relatively positive intravesicular potential with the use of valinomycin and an inwardly-directed K+ gradient, suggesting that biotin transport is most probably an electroneutral process. This suggestion was further supported by studies on biotin transport in the presence of anions of different lipid permeability. The results of this study demonstrate that biotin transport across rat intestinal brush-border membrane is by a carrier-mediated, Na+-dependent and electroneutral process. Furthermore, transport of biotin is higher in the jejunum than the ileum.     

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.     

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.