Stoichiometric studies of the renal outer cortical brush border membraned-glucose transporter

Summary The stoichiometric properties of the renal outer cortical brush-border membraned-glucose transporter are studied. Experiments which establish the glucose/sodium, glucose/phlorizin and phlorizin/sodium stoichiometries are reported. Three independent methods of determining the substrate/activator (glucose/sodium) stoichiometry for coupled transport systems are presented and discussed. One of these, the Static Head Method, is introduced here for the first time. This type of experiment appears to be more generally applicable than the usual procedure of directly measuring the coupled fluxes of substrate and activator to determine stoichiometric coupling ratios. The results presented in this paper demonstrate that the glucose/sodium/phlorizin stoichiometry of the renal outer cortical brush-border membraned-glucose transport system is 111.     

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.     

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.     

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.     

Cytochromes in the rat kidney brush border membrane.

Spectrophotometric studies of the brush border membrane fraction of the rat kidney as compared with those of its mitochondria and microsomes were carried out. Occurrence of cytochromes has been demonstrated in the brush border membranes. Either in the brush border membranes and in the mitochondria evidence for the presence of cytochromes of the types $\text{a}\text{,}\text{b}$ and $\text{c}$ was found, whereas in the microsomes only cytochrome $\text{b}$ was demonstrated.     

Diuretic effects on renal brush border membrane transport and metabolism

Previous studies have indicated that the thiazide diuretics exert effects on proximal electrolyte transport. To determine whether the locus of these effects is at the brush border membrane (BBM) and if renal metabolism is affected, adult female Sprague-Dawley rats were acutely treated with either 1 mg/kg metolazone, 20 mg/kg chlorothiazide followed by a 20 mg/kg/hr maintenance infusion, 10 mg/kg acetazolamide followed by a 10 mg/kg/hr maintenance infusion, or the vehicles only. Administration of these agents resulted in an approximately tenfold increase in sodium excretion. Neither urinary phosphate nor inulin excretion changed significantly in any group. Sodium dependent BBM vesicle phosphate transport was examined at 0.15, 0.5, and 1 and 120 minute incubation periods in the diuretic treated groups and their respective control groups. Decreased uptake was seen in all pre-equilibrium time points in rats treated with metolazone: 0.15 minutes: 221 +/- 24 pmoles/mg protein (pmol/mg prot) in control rats versus (vs) 185 +/- 23 pmoles/mg prot in metolazone-treated animals (P less than .05) ; 0.5 minutes: 463 +/- 54 vs 369 +/- 49 pmol/mg prot (P less than .005); 1 minute: 549 +/- 74 vs 460 +/- 61 pmol/mg prot (P less than .05); no significant difference in phosphate transport was noted at the two hour equilibrium time point. No significant differences in sodium dependent phosphate transport existed between chlorothiazide or acetazolamide treated rats and control animals. Substrate-stimulated renal gluconeogenesis did not differ between metolazone treated and control animals. We therefore conclude that metolazone inhibits phosphate transport through an effect on the BBM and does not affect renal gluconeogenesis in the rat.     

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.     

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.     

Influence of age on duodenal brush border membrane and specific activities of brush border membrane enzymes in Wistar rats.

To examine age-related changes in the morphology of intestinal brush border membrane (BBM; microvilli) and specific activities of intestinal BBM enzymes including alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (gamma-GT), and disacchridase, four groups of Wistar rats were sacrificed at 2.5 wk, 5 wk, 5 mon and 23 mon. In an electron microscopic examination, morphologically a less dense BBM structure in the duodenum of rats aged 23 mon was observed than that of rats aged 5 mon. Specific activity of ALP in the duodenum from 5-mon-old rats was significantly higher than from rats aged 2.5 wk and 23 mon. The mucosal tissues from 5-wk-old rats had significantly higher specific activity of gamma-GT than did tissues from the other ages. In sucrase and maltase specific activities, 5-mon-old rats had higher activities of these enzymes than other age groups, especially 2.5-wk- and 23-mon-old rats. There was also a significant effect of site on intestinal BBM enzyme activities in post-weanling rats. Regional gradients of ALP and gamma-GT along the entire small intestine (duodenum > jejunum > ileum) were remarkable. Disaccharidase activities peaked in the jejunum and declined toward both the duodenum and ileum. Taken together the result obtained here suggested that 5-mon-old rats had the most elevated intestinal function. This result also strongly indicated that the structure of the intestinal BBM and development of intestinal BBM enzymes in Wistar rate were markedly influenced by age during the postnatal period.     

Cyclic nucleotide phosphodiesterases of rabbit renal cortex. Characterization of brush border membrane activities.

Cyclic nucleotide phosphodiesterase activity in brush border membranes, isolated from proximal tubule cells of the rabbit renal cortex, was investigated. Brush border cAMP phosphodiesterase activity was tightly bound to the membrane and was distinguished from the soluble phosphodiesterase activity of the renal cortex cytosol. Multiple forms of the brush border membrane cAMP phosphodiesterase activity, dependent on the concentration of substrate, were found. When assayed with 1 μm or 1 mm cAMP, activities differed in pH optimum, effects of various divalent cations, inhibition by metal ion chelators and reactivation by metals, thermolability, sensitivity to inhibitors and specificity.Renal brush border membranes also possessed cGMP phosphodiesterase activity. cAMP was a relatively poor inhibitor of the hydrolysis of 1 μm cGMP and the hydrolysis of 1 μm cAMP was virtually insensitive to cGMP. These findings suggest that the low substrate concentration-dependent cAMP phosphodiesterase was distinct from the low substrate concentration-dependent cGMP phosphodiesterase.Heat-stable effectors of phosphodiesterase activity were found in the renal cortex. One effector activated soluble cAMP phosphodiesterase. Activation was decreased by EGTA, enhanced by Ca²⁺ and diminished by preincubating the effector with proteolytic enzymes. The other heat-stable effector inhibited brush border membrane phosphodiesterase activity. Inhibition was unaffected by metal ions, unaffected by preincubating the effector with proteolytic enzymes, but diminished by preincubation with phospholipase C and neuraminidase.It is suggested that changes in the activity of the enzyme (or enzymes), which in turn controls, in part, the effective concentration of cAMP at its site (or sites) of action in the renal cell, may be significant in regulating hormonal-dependent transport in the proximal tubule.     

Fractionation of renal brush border membrane proteins with Triton X-114 phase partitioning.

Analysis of brush border membrane proteins by gel electrophoresis has revealed a complex polypeptide composition. We have investigated the use of Triton X-114 phase partitioning to fractionate such proteins on the basis of their degree of hydrophobicity. Each of the fractions was composed of a complex but distinct set of proteins. Most proteins were solubilized by Triton X-114 and partitioned into the detergent-poor fraction. Trehalase, gamma-glutamyl transpeptidase, and leucine aminopeptidase were well solubilized (greater than 80%) and enriched 5.1-, 3.9-, and 2.5-fold in the detergent-rich fraction. In contrast, alkaline phosphatase and 5'-nucleotidase were poorly solubilized. The specific activities of these enzymes were increased 2.7- and 2.3-fold in the insoluble protein fraction. Maltase was almost completely solubilized and partitioned into the detergent-poor fraction with a small enrichment factor (1.3). These results suggest that Triton X-114 phase partitioning could be useful as a first step in the purification of many brush border membrane proteins.     

Dipeptide transport in isolated intestinal brush border membrane.

Transport of glycyl-L-leucine into isolated brush border membrane vesicles was studied. On the basis of the following observations it was postulated that glycyl-L-leucine was transported intact by a specific dipeptide mechanism. (1) The differing time course and Na-+ stimulation of glycine, L-leucine and glycyl-L-leucine. (2) The failure of glycine and L-leucine to inhibit glycyl-L-leucine transport. (3) Initial presence of dipeptide within the vesicle. (4) Inhibition of glycyl-L-leucine uptake by other dipeptides. (5) The occurrence of accelerated amino acid uptake in the presence of the dipeptide.     

Purification of brush border membrane vesicles from rat renal cortex by size-exclusion chromatography.

Size-exclusion chromatography with controlled pore glass (CPG) was used in the further purification of renal brush border membrane vesicles (BBMV) isolated by the Ca precipitation method. The BBMV obtained had an almost spherical shape and their average diameter was about 95 nm in isotonic solution. The specific activities of alkaline phosphate and leucine aminopeptidase in the BBMV preparation were increased 18- and 17-fold, respectively, over those in the crude homogenate. The uptake of D-glucose by the purified BBMV in the presence of a sodium gradient reached 8.53 nmol/mg protein at 20 s. These results indicate that CPG chromatography is suitable procedure by which to obtain purified renal BBMV of homogenous size and with high specific marker enzyme activity for use in the study of membrane transport.     

Phenylalanine uptake in isolated renal brush border vesicles.

The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up L-phenylalanine by an Na+-dependent, saturable transport system. The apparent affinity of the transport system for L-phenylalanine is 6.1 mM at 100 mM Na+ and for Na+ 13mM at 1 mM L-phenylalanine. Reduction of the Na+ concentration reduces the apparent affinity of the transport system for L-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K+ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of L-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na+ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na+ and on the electrical potential difference. The exit of L-phenylalanine across the basolateral plasma membranes is Na+-independent and probably involves facilitated diffusion.