Decreased transport of D-glucose and L-alanine across brush-border membrane vesicles from small intestine of rats treated with mitomycin C

To elucidate the mechanisms underlying the dysfunctions of intestinal absorption induced by antitumor drugs, the effect of pretreatment with mitomycin C on sodium gradient-dependent D-glucose and L-alanine transports was studied in rat brush-border membrane vesicles. 24, 48, 96, or 120 h following a single intravenous injection of mitomycin C, brush-border membrane vesicles were prepared from rat small-intestines. The uptake of D-glucose and L-alanine was shown to be Na+ gradient-dependent even in the case of vesicles obtained from mitomycin C-treated rats, but uptake rates measured at 15 s and magnitude of overshooting effect in uptake of both solutes were decreased in vesicles maximally from 48 h mitomycin C-treated rats. The rate of D-glucose uptake calculated at 15 s recovered to the control level in vesicles prepared at 96 h and 120 h after mitomycin C-treatment, indicating that the effect of mitomycin C on Na+ gradient-dependent D-glucose transport would be fully reversible. Tracer exchange experiments under Na+ and D-glucose equilibrated conditions indicated that the Na+/D-glucose transporters were similarly operative in the vesicles from control and 48 h mitomycin C-treated rats. Rates of 22Na+ uptake measured at 15 s in vesicles from 48 h mitomycin C-treated rats, however, were increased. The increased permeability to Na+ might bring about a more rapid dissipation of the Na+ gradient in these vesicles and this would secondarily cause the decrease in Na+-dependent D-glucose uptake in vesicles from mitomycin C-treated rats.     

Na+-coupled D-glucose uptake and membrane order of enterocyte brush border membrane vesicles, under the effect of a series of N-phenylcarbamates

The importance of the hydrophobic effect of exogenous substances and of modifications of membrane order on D-glucose uptake are still poorly defined. Our results show that the concentrative Na+ -coupled D-glucose uptake of rat enterocyte brush border membrane vesicles is inhibited by N-phenylcarbamates increase the membrane order. However, since the concentrations required for membrane order increase are much greater than those active on D-glucose uptake, the effects on lipid order cannot be responsible for the inhibition of D-glucose uptake. Measurements of D-glucose uptake under conditions of Na+ equilibrium show that these carbamates do not act directly on the carrier but indirectly by favouring the dissipation of the Na+ gradient.     

The Na+ gradient-dependent transport of D-glucose in renal brush border membranes.

The Na+-dependent transport of D-glucose was studied in brush border membrane vesicles isolated from the rabbit renal cortex. The presence of a Na+ gradient between the external incubation medium and the intravesicular medium induced a marked stimulation of D-glucose uptake. Accumulation of the sugar in the vesicles reached a maximum and then decreased, indicating efflux. The final level of uptake of the sugar in the presence of the Na+ gradient was identical with that attained in the absence of the gradient, suggesting that equilibrium was established. At the peak of the overshoot the uptake of D-glucose was more than 10-fold the equilibrium value. These results suggest that the imposition of a large extravesicular to intravesicular gradient of Na+ effects the transient movement of D-glucose into renal brush border membranes against its concentration gradient. The stimulation of D-glucose uptake into the membranes was specific for Na+. The rate of uptake was enhanced with increased concentration of Na+. Increasing Na+ in the external medium lowered the apparent Km for D-glucose. The Na+ gradient effect on D-glucose transport was dissected into a stimulatory effect when Na+ and sugar were on the same side of the membrane (cis stimulation) and an inhibitory effect when Na+ and sugar were on opposite sides of the membrane (trans inhibition). The uptake of D-glucose, at a given concentration of sugar, reflected the sum of the contributions from a Na+-dependent transport system and a Na+-independent system. The relative stimulation of D-glucose uptake by Na+ decreased as the sugar concentration increased. It is suggested, however, that at physiological concentrations of D-glucose the asymmetry of Na+ across the brush border membrane might fully account for uphill D-glucose transport. The physiological significance of the findings is enhanced additionally by observations that the Na+-dependent D-glucose transport system in the membranes in vitro possessed the sugar specificities and higg phlorizin sensitivity characteristic of more intact preparations. These results provide strong experimental evidence for the role of Na+ in transporting D-glucose across the renal proximal tubule luminal membrane.     

Biotin uptake mechanisms in brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex

Biotin transport was studied using brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex. An inwardly directed Na+ gradient stimulated biotin uptake into brush-border membrane vesicles and a transient accumulation of the anion against its concentration gradient was observed. In contrast, uptake of biotin by basolateral membrane vesicles was found to be Na+-gradient insensitive. Generation of a negative intravesicular potential by valinomycin-induced K+ diffusion potentials or by the presence of Na+ salts of anions of different permeabilities enhanced biotin uptake by brush-border membrane vesicles, suggesting an electrogenic mechanism. The Na+ gradient-dependent uptake of biotin into brush-border membrane vesicles was saturable with an apparent Km of 28 microM. The Na+-dependent uptake of tracer biotin was significantly inhibited by 50 microM biotin, and thioctic acid but not by 50 microM L-lactate, D-glucose, or succinate. Finally, the existence in both types of membrane vesicles of a H+/biotin- cotransport system could not be demonstrated. These results are consistent with a model for biotin reabsorption in which the Na+/biotin- cotransporter in luminal membranes provides the driving force for uphill transport of this vitamin.     

Factors affecting the transport of beta-amino acids in rat renal brush-border membrane vesicles. The role of external chloride

The effect of a variety of ions and other solutes on the accumulation of the beta-amino acid, taurine, was examined in rat renal brush-border membrane vesicles. Initial taurine uptake (15 and 30 s) is sodium-dependent with a typical overshoot. This Na+ effect was confirmed by exchange diffusion and gramicidin inhibition of taurine uptake. External K+ or Li+ do not increase taurine accumulation more than Na+-free mannitol, except that the combination of external K+ and Na+ in the presence of nigericin enhances uptake. Of all anions tested, including more permeant (SCN- and NO3-) or less permeant (SO4(2-)), chloride supported taurine accumulation to a significantly greater degree. Preloading vesicles with choline chloride reduced taurine uptake, suggesting that external Cl- stimulates uptake. Since this choline effect could be related to volume change, due to the slow diffusion of choline into vesicles, brush-border membrane vesicles were pre-incubated with LiCl, LiNO3 and LiSO4. Internal LiCl, regardless of the final Na+ anion mixture, reduced initial rate (15 and 60 s) and peak (360 s) taurine uptake. Internal LiNO3 or LiSO4 with external NaCl resulted in similar or higher values of uptake at 15, 60 and 360 s, indicating a role for external Cl- in taurine uptake in addition to Na+ effect. Although uptake by vesicles is greatest at pH 8.0 and inhibited at acidic pH values (pH less than 7.0), an externally directed H+ gradient does not influence uptake. Similarly, amiloride, an inhibitor of the Na+/H+ antiporter, had no influence on taurine accumulation over a wide variety of concentrations or at low Na+ concentrations. Taurine uptake is blocked only by other beta-amino acids and in a competitive fashion. D-Glucose and p-aminohippurate at high concentrations (greater than 10(-3) M) reduce taurine uptake, possibly by competing for sodium ions, although gramicidin added in the presence of D-glucose inhibits taurine uptake even further. These studies more clearly define the nature of the renal beta-amino acid transport system in brush-border vesicles and indicate a role for external Cl- in this uptake system.     

Brush border membrane vesicles formed from human duodenal biopsies exhibit Na+-dependent concentrative L-leucine and D-glucose uptake

The human duodenum actively transports L-leucine and D-glucose under Na+ gradient conditions as demonstrated by uptake studies using brush border membrane vesicles from organ donor duodenum. Brush border membrane vesicles formed from peroral duodenal biopsies likewise demonstrate Na+ dependent concentrative uptake of D-glucose and L-leucine. This is the first demonstration of active transport processes in human duodenum. A simple microvesiculation method to form these vesicles is described as well as its potential application to clinical medicine in studying diseases of defective intestinal transport.     

The mechanism of decreased Na+-dependent D-glucose transport in brush-border membrane vesicles from rabbit kidneys with experimental Fanconi syndrome

In our previous paper (Yanase, M. et al. (1983) Biochim. Biophys. Acta 733, 95-101) we reported that the Na+-dependent D-glucose uptake into brush-border membrane vesicles is decreased in rabbits with experimental Fanconi syndrome (induced by anhydro-4-epitetracycline). In the present paper we investigate the mechanism underlying this decrease. D-Glucose is taken up into the osmotically active space in anhydro-4-epitetracycline-treated brush-border membrane vesicles and exhibits the same distribution volume and the same degree of nonspecific binding and trapping as in control brush-border membrane vesicles. The passive permeability properties of control and anhydro-4-epitetracycline-treated brush-border membrane vesicles are shown to be the same as measured by the time-dependence of L-glucose efflux from brush-border membrane vesicles. D-Glucose flux was measured by the equilibrium exchange procedure at constant external and internal Na+ concentrations and zero potential. Kinetic analyses of Na+-dependent D-glucose flux indicate that Vmax in anhydro-4-epitetracycline-treated brush-border membrane vesicles (79.3 +/- 7.6 nmol/min per mg protein) is significantly smaller than in control brush-border membrane vesicles (141.3 +/- 9.9 nmol/min per mg protein), while the Km values in the two cases are not different from each other (22.3 +/- 0.9 and 27.4 +/- 1.8 mM, respectively). These results suggest that Na+-dependent D-glucose carriers per se are affected by anhydro-4-epitetracycline, and that this disorder is an important underlying mechanism in the decreased Na+-dependent D-glucose uptake into anhydro-4-epitetracycline-treated brush-border membrane vesicles.     

Hydrogen ion-coupled transport of D-glucose by phlorizin-sensitive sugar carrier in intestinal brush-border membranes

In rabbit intestinal brush-border membrane vesicles, Na+-independent D-glucose uptake in the presence of an inside-negative transmembrane potential was found to be stimulated by an imposed pH gradient. Na+-independent, pH-dependent and phlorizin-sensitive D-glucose-evoked potentials could be recorded from isolated toad intestine. The obtained data suggest that phlorizin-sensitive D-glucose carriers of intestinal brush-border membrane can interact with H+ when Na+ is absent.     

Effect of 25-hydroxyvitamin D3 on Na+-dependent D-glucose transport in rachitic rats

We have previously reported that feeding rats on Steenbock and Black's rickets-inducing diet, deficient in vitamin D and with an altered Ca/P ratio, leads to metabolic consequences and a marked decrease of Na+-dependent D-glucose uptake at the jejunum-ileum level. To clarify the relationship between experimental rickets and D-glucose uptake, 25-hydroxyvitamin D3 (25-OH-D3) was given to rats fed on the rickets inducing diet. In the jejunum-ileum of these animals Na+-dependent D-glucose uptake returned to the values of the controls while the decrease in D-glucose uptake in the brush-border membrane vesicles prepared from kidney cortex of rachitic animals was not corrected by the administration of 25-OH-D3.     

1,25-Dihydroxycholecalciferol-related Na+/D-glucose transport in brush-border membrane vesicles from embryonic chick jejunum. Modulation by triiodothyronine

1,25-Dihydroxycholecalciferol, when present at and above 10 nM in an organ-culture system of embryonic chick jejunum, approximately doubled the rate of Na(+)-gradient-driven D-glucose uptake by brush-border membrane vesicles, but had no effect on Na(+)-independent D-glucose transfer. The sterol also had no effect on Na+ influx along an outside/inside Na+ gradient ([Na+]o = 100 mM; [Na+]i = 0 mM). This renders it unlikely that in embryonic intestine, calcitriol raises Na(+)-dependent D-glucose transport through changes in the electrochemical Na+ gradient. D-[U-14C]Glucose tracer exchange, measured under voltage-clamp condition at Na+/D-glucose equilibrium, revealed that addition of calcitriol to the culture medium approximately doubled the activity of the Na+/D-glucose transporter in the brush-border membrane. This was also reflected by an corresponding increase in the maximal velocity of the transfer process. Increased [3H]phlorizin binding after calcitriol treatment suggests that the steroid hormone activates Na+/D-glucose transport through increasing the number of carrier molecules in the brush-border membrane. 10 nM triiodothyronine, which by itself has no effect on Na(+)-dependent D-glucose transport, potentiated the effect of 1,25-dihydroxycholecalciferol such that in the presence of both hormones, Na+/D-glucose-carrier activity was increased fourfold above control levels.     

Insulin regulates Na+/glucose cotransporter activity in rat small intestine

In order to examine the involvement of insulin in the activity of Na+/glucose cotransporter in rat small intestine, we compared Na(+)-dependent uptake of D-glucose by brush-border membrane vesicles prepared from control, streptozotocin-induced diabetic, insulin-treated diabetic and starved diabetic rats. In four groups, the uptake of D-glucose showed a transient overshoot in the presence of Na+ gradient between medium and vesicles (medium greater than vesicles). The overshoot magnitude was increased (1.8-fold of controls) in diabetic brush border membrane vesicles and recovered to the control level by the treatment of diabetic rats with insulin. In contrast, increased uptake of D-glucose in diabetic rats was not recovered by the starvation of diabetic rats although the blood glucose level was the same as that of controls. Furthermore, we attempted to examine phlorizin binding activities among four groups. Scatchard analysis indicated that phlorizin binding to diabetic brush border membrane vesicles was increased (1.6-fold of controls) without a change of the affinity for phlorizin as compared with controls. Increased binding of phlorizin to diabetic brush border membrane vesicles was also recovered to the control level by the treatment of diabetic rats with insulin, but not by starvation. These results suggested that the increased activity of Na+/glucose cotransporter in diabetic rats was due to the increase of the number of cotransporter and that intestinal cotransporter was physiologically controlled by insulin, but not by blood glucose levels.     

D-glucose uptake in intestinal brush-border membrane vesicles of rachitic rats

We have previously reported the metabolic consequences of feeding rats Steenbock and Black's rickets-inducing diet, deficient in vitamin D and with an altered Ca/P ratio. Using isolated brush-border membrane vesicles prepared from the jejunum, ileum and duodenum of control and rachitic rats, we have demonstrated a marked decrease of Na+-dependent D-glucose uptake at jejunum-ileum level of rachitic rats. At duodenum level Na+-dependent D-glucose transport was not influenced by rickets. A lack of any significant difference between the two animal groups was observed studying the facilitated transport of D-glucose, the diffusion of L-glucose and the Na+-dependent uptake of phenylalanine and aspartate.     

Regulation of amino acid and glucose transport activity expressed in isolated membranes from untransformed and SV 40-transformed mouse fibroblasts.

Membrane vesicles isolated from untransformed Balb/c and Swiss mouse fibroblasts and their SV 40-transformed derivatives were shown to catalyze carrier-mediated, intravesicular uptake of alpha-aminoisobutyric acid and D-glucose. Concentrative uptake of alpha-aminoisobutyric acid required the presence of a Na+-gradient (external greater than internal) and could occur independently of endogenous (Na+ + K+)ATPase activity. A K+ diffusion gradient (internal greater than external) in the presence of valinomycin, or the addition of the Na+ salt of a highly permeant anion, conditions expected to create an interior-negative membrane potential stimulated Na+-gradient-dependent uptake, suggesting this process is electrogenic. D-Glucose uptake was nonconcentrative and did not require ion gradients or metabolic conversion. Na+ gradient-dependent transport of alpha-aminoisobutyric acid was reduced both in initial rate and extent of uptake in vesicles from confluent untransformed cells and increased in those from SV 40-transformed cells, compared with activities observed in vesicles from proliferating untransformed cells. No changes in D-glucose carrier activity were observed when assayed at low glucose concentrations.     

Radiation-inactivation studies on brush-border-membrane vesicles. General considerations, and application to the glucose and phosphate carriers.

Radiation-inactivation studies were performed on brush-border-membrane vesicles purified from rat kidney cortex. No alteration of the structural integrity of the vesicles was apparent in electron micrographs of irradiated and unirradiated vesicles. The size distributions of the vesicles were also similar for both populations. The molecular sizes of two-brush-border-membrane enzymes, alkaline phosphatase and 5'-nucleotidase, estimated by the radiation-inactivation technique, were 104800 +/- 3500 and 89,400 +/- 1800 Da respectively. Polyacrylamide-gel-electrophoresis patterns of membrane proteins remained unaltered by the radiation treatment, except in the region of higher-molecular-mass proteins, where destruction of the proteins was visible. The molecular size of two of these proteins was estimated from their mobilities in polyacrylamide gels and was similar to the target size, estimated from densitometric scanning of the gel. Intravesicular volume, estimated by the uptake of D-glucose at equilibrium, was unaffected by irradiation. Uptake of Na+, D-glucose and phosphate were measured in initial-rate conditions to avoid artifacts arising from a decrease in the driving force caused by a modification of membrane permeability. Na+-independent D-glucose and phosphate uptakes were totally unaffected in the dose range used (0-9 Mrad). The Na+-dependent uptake of D-glucose was studied in irradiated vesicles, and the molecular size of the transporter was found to be 288,000 Da. The size of the Na+-dependent phosphate carrier was also estimated, and a value of 234,000 Da was obtained.     

The asymmetric effect of lanthanides on Na+-gradient-dependent Ca2+ transport in synaptic plasma membrane vesicles

Lanthanides (La3+, Pr3+ and Tb3+) inhibit Na+-gradient-dependent Ca2+ influx into synaptic plasma membrane vesicles. 50% inhibition is obtained by 7 microM lanthanide concentration. The inhibition of the Na+-gradient-dependent Ca2+ uptake exhibits competitive kinetic behaviour. The apparent Km of the Ca2+ influx is increased from 50 microM in the absence of lanthanides to 118 microM in the presence of La3+, 170 microM in the presence of Pr3+ and 130 microM in the presence of Tb3+. The maximal reaction velocity is not altered (8.35 nmol Ca2+ transported per mg protein per min in the absence of lanthanides and 8.16 nmol/mg per min in the presence of lanthanides). Lanthanides also inhibited Na+-gradient-dependent Ca2+ efflux from synaptic plasma membrane vesicles that were preloaded with Ca2+ in a Na+-gradient-dependent manner. Introduction of La3+ into the interior of the synaptic plasma membrane vesicles by rapid freezing of the vesicles in liquid N2 and slow thawing had no effect on either Na+-gradient-dependent Ca2+ influx or efflux. Synaptic plasma membrane vesicles can be preloaded with Ca2+ also in an ATP-dependent manner. This form of Ca2+ uptake is also inhibited by La3+ though at higher concentrations than the Na+-gradient-dependent Ca2+ uptake. Na+-gradient-dependent efflux from synaptic plasma membrane vesicles preloaded in an ATP-dependent fashion ('inside-out' vesicles) unlike efflux from synaptic plasma membrane vesicles preloaded in a Na+-gradient-dependent manner was not inhibited by La3+. These findings suggest that the inhibition by La3+ is manifested asymmetrically on both sides of the synaptic plasma membrane. Lanthanides are probably not transported via the Na+-Ca2+ exchanger since Tb3+ entry measured by fluorescence of Tb3+-dipicolinic acid complex formation occurred at high Tb3+ concentrations only (1.5 mM or above) and was not Na+-gradient dependent.     

Sugar transport by renal plasma membrane vesicles. Characterization of the systems in the brush-border microvilli and basal-lateral plasma membranes.

Uptake studies of D- and L-glucose were performed on vesicles derived from brush-border and basal-lateral membranes. The uptake of the sugars into the vesicles was osmotically sensitive and independent of glucose metabolism. In brush-border vesicles D-glucose but not L-glucose transport was Na+ -dependent, was inhibited by phlorizin, and showed a transitory vesicle/medium ratio greater than 1, in the presence of an initial Na+ gradient. Basal-lateral membranes take up D-glucose faster than L-glucose, but the D-glucose uptake is significantly less sensitive to sodium removal and only moderately inhibited by phlorizin as compared to the brush-border fraction.     

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.     

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.     

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.     

Gentamicin inhibits Na+-dependent D-glucose transport in rabbit kidney brush-border membrane vesicles

We studied the effect of gentamicin on Na+-dependent D-glucose transport into brush-border membrane vesicles isolated from rabbit kidney outer cortex (early proximal tubule) and outer medulla (late proximal tubule) in vitro. We found the same osmotically active space and nonspecific binding between control and gentamicin-treated brush-border membrane vesicles. There was no difference in the passive permeability properties between control and gentamicin-treated brush-border membrane vesicles. Kinetic analyses of D-glucose transport into 1 mM gentamicin-treated brush-border membrane vesicles demonstrated that gentamicin decreased Vmax in the outer cortical preparation, while it did not affect Vmax in the outer medullary preparation. With regard to Km, there was no effect of gentamicin in any vesicle preparation. When brush-border membrane vesicles were incubated with higher concentrations of gentamicin, Na+-dependent D-glucose transport was inhibited dose-dependently in both outer cortical and outer medullary preparations. Dixon plots yield inhibition constant Ki = 4 mM in the outer cortical preparation and Ki = 7 mM in the outer medullary preparation. These results indicate that the Na+-dependent D-glucose transport system in early proximal tubule is more vulnerable to gentamicin toxicity than that in late proximal tubule.