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.     

1-O-n-octyl-beta-D-glucopyranoside as a competitive inhibitor of Na+-dependent D-glucose cotransporter in the small intestine brush-border membrane

1-O-n-Octyl-beta-D-glucopyranoside is a competitive inhibitor of the Na+-dependent D-glucose uptake into rabbit, rat and human intestinal brush-border membrane vesicles. The lack of effect on the equilibrium uptake demonstrates that the detergent does not act by rupturing the vesicles; no membrane leakiness was apparent at the concentrations of octylglucopyranoside used, since D-glucose uptake is not inhibited even in the absence of the Na+ gradient (in K+ solution). There is a competitive interaction between octylglucopyranoside and D-glucose, as shown by Dixon and by Hunter and Down plots. The selectivity of the detergent effect is confirmed by its modest influence on amino acid uptake.     

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.     

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.     

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.     

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.     

Kinetics of D-glucose transport across the intestinal brush-border membrane of the cat

1. D-glucose transport across the intestinal brush-border membrane of the cat, a carnivorous animal, was investigated using isolated brush-border membrane vesicles (BBMV). Kinetic experiments were performed under zero-trans conditions (initial [Na+]in and [Gluc]in = O) with the transmembrane electrical potential difference clamped to zero. 2. D-glucose uptake by the BBMV was strongly stimulated by an inwardly directed Na+-gradient. Uptake under Na+-free conditions seemed to occur by simple diffusion. 3. The apparent kinetic constants (Vmax, Km) of Na+-dependent D-glucose transport were computed by forcing initial uptake rates at 0.002-10.0 mmol/l D-glucose to either a Michaelis-Menten type equation with a single or with two carrier-mediated components. 4. Best fit of the experimental data was obtained with the two-component model indicating the existence of two Na+-dependent carrier-mediated mechanisms. System 1 and system 2 differ with respect to the transport velocity as well as the substrate affinity constants with Vmax being 2.5-fold and Km being 5-fold higher for system 1 compared with system 2.     

Differences in neutral amino acid and glucose transport between brush border and basolateral plasma membrane of intestinal epithelial cells.

A comparison of L-valine and D-glucose transport was carried out with vesicles of plasma membrane isolated either from the luminal (brush border) or from the contra-luminal (basolateral) region of small intestinal epithelial cells. The existence of transport systems for both non-electrolytes was demonstrated by stereospecificity and saturability of uptake, as well as tracer coupling. Transport of L-valine and D-glucose differs markedly in the two types of plasma membrane with respect to stimulation by Na+. The presence of Na+ stimulated initial L-valine and D-glucose uptake in brush border, but not in basolateral membrane. Moreover, an electro-chemical Na+ gradient, oriented with the lower potential on the inside, supported accumulation of the non-electrolytes above medium concentration only in the brush border membrane. L-Valine and D-glucose transport also were saturated at lower concentrations in brush border (10-20 mM) than in basolateral plasma membranes (30-50 mM). A third difference between the two membranes was found in the effectiveness of known inhibitors of D-glucose transport. In brush border membranes phlorizin was more potent than phloretin and 2', 3', 4'-trihydroxy-4-methoxy chalcone and cytochalasin B did not inhibit at all. In contrast, with the basolateral plasma membranes the order of potency was changed to phloretin = 2',3',4'-trihydroxy-4-methoxy chalcone greater than cytochalasin B greater than phlorizin. These results indicate the presence of different types of transport systems for monosaccharides and neutral amino acids in the luminal and contra-luminal region of the plasma membrane. Active transepithelial transport can be explained on the basis of the different properties of the non-electrolyte transport systems in the two cellular regions and an electro-chemical Na+ gradient that is dependent on cellular metabolism.     

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.     

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.     

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.     

Enhancement of glucose transport in small intestinal brush border membrane of retinol administered rat

Retinylpalmitate (200 IU/kg body weight) was administered intraperitoneally to rats once daily for 4 days. Brush border membrane vesicles (BBMVs) were prepared from small intestinal epithelium cells from along the crypt-villus axis. D-glucose uptake by BBMVs was examined under the inwardly directed Na+ gradient. The D-glucose uptake by BBMVs from the villus-tip and mid-villus cells of retinylpalmitate treated rats was significantly larger than that of control (corn oil treated) rats, respectively. Thus, retinol treatment of rats promoted the D-glucose transport in small intestinal brush border membrane. Interestingly, the enhancement of D-glucose transport was more prominent in villus-tip and mid-villus than in lower villus.     

Decreased D-glucose transport across renal brush-border membrane vesicles from streptozotocin-induced diabetic rats

The uptake of Na(+)-dependent D-glucose by renal brush-border membrane vesicles (BBMV) isolated from streptozotocin-induced diabetic rats was decreased as compared with controls. Since a Vmax of 4.8 nmol/mg protein per 30 s in diabetic BBMV was significantly decreased as compared with that of controls (Vmax = 7.0 nmol/mg protein per 30 s) without changing an apparent affinity for D-glucose, the decrease in the Na(+)-dependent D-glucose uptake in diabetic rats is likely to be due to the reduction in the number of the transporter. These results are also confirmed by the binding study of [3H]phlorizin to diabetic BBMV. When the blood glucose level is lowered in diabetic rats by both the treatment with insulin and starvation, the decreased Na(+)-dependent D-glucose uptake is returned to control level. These results suggest that Na(+)-dependent D-glucose reabsorption through the apical membrane in proximal tubular kidney cells is dynamically regulated by the change in blood glucose level.     

Temperature sensitivity and substrate specificity of two distinct Na+-activated D-glucose transport systems in guinea pig jejunal brush border membrane vesicles

D-Glucose transport was studied with isolated brush border membrane vesicles from guinea pig jejunum. Saturation curves were carried out at either 25 or 35 degrees C in buffers containing Na+, Li+, K+ (100 mM chloride salt), or sorbitol (200 mM). Uncorrected uptake rates were fitted by nonlinear regression analysis to an equation involving one diffusional and two saturable terms. In the presence of Na+ at 35 degrees C, two saturable systems (Km = 0.4 and 24 mM, respectively) were evident, as well as a diffusion component quantitatively identical with that measured with L-glucose in separate experiments. In contrast, at 25 degrees C only one saturable system was apparent (Km = 1.2 mM): the second exhibited diffusion-like kinetics. In the presence of Na+ at 35 degrees C, D-glucose uptake was fully inhibited by both D-glucose and D-galactose, whereas alpha-methylglucoside gave kinetics of partial inhibition. We conclude that in the presence of Na+ there are at least two distinct D-glucose transport systems: 1) System I, a low temperature-sensitive system, fully inhibited by D-glucose, D-galactose, and alpha-methylglucoside; we identify it as the "classical" D-glucose/Na+ cotransport system, insensitive to inhibition by cytochalasin B and obligatorily dependent on Na+; and 2) System II, a high temperature-sensitive system where D-glucose and D-galactose inhibit but alpha-methylglucoside is inert. Its cation specificity is unclear but it appears to be sensitive to cytochalasin B inhibition. When Li+ or K+ substituted for Na+, only one transport system was apparent. The Li+-activated transport was: independent of the incubation temperature; inhibited by D-glucose and D-galactose but not by alpha-methylglucoside, 2-deoxy-D-glucose, D-mannose, and D-xylose; and sensitive to cytochalasin B inhibition. The exact nature of the system (or systems) involved in D-glucose transport in the absence of sodium remains to be established.     

Characteristics of D-galactose transport systems by luminal membrane vesicles from rabbit kidney

The characteristics of renal transport of D-galactose by luminal membrane vesicles from either whole cortex, pars recta or pars convoluta of rabbit proximal tubule were investigated by a spectrophotometric method using a potential-sensitive carbocyanine dye. Uptake of D-galactose by luminal membrane vesicles prepared from whole cortex was carried out by an Na+-dependent and electrogenic process. Eadie-Hofstee analysis of saturation-kinetic data suggested the presence of multiple transport systems in vesicles from whole cortex for the uptake of D-galactose. Tubular localization of the transport systems was studied by the use of vesicles derived from pars recta and pars convoluta. In pars recta, Na+-dependent transport of D-galactose and D-glucose occurred by means of a high-affinity system (half-saturation: D-galactose, 0.15 +/- 0.02 mM; D-glucose, 0.13 +/- 0.02 mM). These results indicated that the "carrier' responsible for the uptake of these hexoses does not discriminate between the steric position of the C-4 hydroxyl group of these two isomers. This is further confirmed by competition experiments, which showed that D-galactose and D-glucose are taken up by the same and equal affinity transport system by these vesicle preparations. Uptake of D-galactose and D-glucose by luminal membrane vesicles isolated from pars convoluta was mediated by a low-affinity common transport system (half-saturation: D-galactose, 15 +/- 2 mM; D-glucose, 2.5 +/- 0.5 mM). These findings strongly suggested that the "carrier' involved in the transport of monosaccharides in vesicles from pars convoluta is specific for the steric position of the C-4 hydroxyl group of these sugars and presumably interacts only with D-glucose at normal physiological concentration.     

Towards the mechanism of altered nutrients uptake in renal brush border membrane vesicles from pyelonephritic rats

Affinity constant (Km) of D-glucose, L-alanine, L-aspartate, L-lysine, L-proline and nutrients coupled Na+ were determined in renal brush border membrane vesicles prepared from control and pyelonephritic rats. The Km of D-glucose, amino acids and nutrients coupled Na+ was noted to be significantly increased (p less than 0.001) in experimental animals. The Vmax of D-glucose and amino acids was determined at different concentrations of nutrients keeping extravesicular Na+ constant or at different concentrations of extravesicular Na+ keeping nutrient concentration constant. In the experimental rats the Vmax decreased significantly (p less than 0.01) when compared to control. The increased Km and decreased Vmax may be one of the underlying mechanism leading to decrease in the uptake of D-glucose and amino acids.     

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.     

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.     

Effect of glucose and pyruvate metabolism on membrane potential in synaptosomes

Fluorescence changes of rhodamine 6G in synaptosomal suspension, which are correlated to changes in membrane potential in synaptosomes, were measured in the presence of various monosaccharides and organic acids. Addition of D-glucose, D-mannose, pyruvate and L-lactate hyperpolarized the membrane potential, whereas D-fructose, L-glucose, D-galactose, citrate, succinate and L-glutamate were without effect on the membrane potential. Hyperpolarization induced by D-glucose was inhibited by cytochalasin B, phloretin, iodoacetate, F- and 2-deoxy-D-glucose, but not inhibited by oligomycin or phlorizin. On the other hand, hyperpolarization induced by pyruvate was inhibited by alpha-cyanocinnamate or phloretin, but not inhibited by cytochalasin B or F-. Elimination of Na+ in physiological saline depressed hyperpolarization of membrane potential induced by addition of D-glucose, L-lactate or pyruvate. These results suggest that the activity of (Na+ + K+)-ATPase in plasma membranes of synaptosomes is increased by ATP formed by glycolysis, and that the accumulated K+ in synaptosomes hyperpolarizes the membrane potential.     

In vitro effect of ethanol on sodium and glucose transport in rabbit renal brush border membrane vesicles.

The effect of ethanol on sodium and glucose transport in rabbit renal brush border membrane vesicles was examined. When membrane vesicles were preincubated in the presence of ethanol the sodium-dependent D-glucose uptake was significantly inhibited. This effect, as suggested by O'Neill et al. (1986) FEBS Lett. 194, 183-188, may be due to a faster collapse of the Na+ gradient. As a matter of fact, the amiloride-insensitive sodium pathway was increased by ethanol in our brush border membrane preparation. However, sodium/D-glucose cotransport was inhibited by ethanol, although to a lesser degree, also in the absence of a sodium gradient. In addition, ethanol inhibited glucose-dependent sodium uptake, suggesting that a direct interaction with the translocator was involved. This conclusion was also supported by kinetic measurements showing a decrease of Vmax and an increase in Km for glucose in membrane vesicles treated with ethanol. Moreover, ethanol influenced the interaction of phlorizin with the cotransporter: uptake experiments performed in the presence of the two inhibitors demonstrated that phlorizin and ethanol behave as not mutually exclusive inhibitors of D-glucose transport. These data indicate that in rabbit renal brush border membranes ethanol not only affects the 'passive pathway', i.e. the sodium permeability, but it also directly interferes with carrier functions.