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.     

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.     

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.     

Glucose uptake into plasma membrane vesicles from the maternal surface of human placenta

Summary Glucose uptake into plasma membrane vesicles from the maternal surface of the human placenta was measured with the Millipore filtration technique. Uptake ofd-glucose was dependent on the osmolarity of the incubation medium surrounding the vesicles. Uptake ofd-glucose exceeded that ofl-glucose. The uptake ofd-glucose was not enhanced by placing 100mm NaCl or NaSCN in the medium outside the vesicles (none inside) at the onset of uptake determinations.d-glucose transport was inhibited by cytochalasin B; phloretin, phlorizin, and 1-fluoro-2,4-dinitrobenzene.d-glucose uptake was inhibited by 2-deoxy-d-glucose, 3-O-methyl-d-glucose and to a lesser extent byd-galactose. It was not inhibited by -methyl-d-glucoside. Cytochalasin B binding to the vesicles was 30% inhibited in the presence of 80mm d-glucose. The results indicate that the system for facilitated transport ofd-glucose at the maternal face of the placenta is distinctly different from that on the brush-border membrane of intestine or renal tubule and more closely resembles that of human erythrocyte.     

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.     

Sodium/proton antiport in brush-border-membrane vesicles isolated from rat small intestine and kidney.

Studies on proton and Na+ transport by isolated intestinal and renal brush-border-membrane vesicles were carried out to test for the presence of an Na+/H+-exchange system. Proton transport was evaluated as proton transfer from the intravesicular space to the incubation medium by monitoring pH changes in the membrane suspension induced by sudden addition of cations. Na+ transport was determined as Na+ uptake into the vesicles by filtration technique. A sudden addition of sodium salts (but not choline) to the membrane suspension provokes an acidification of the incubation medium which is abolished by the addition of 0.5% Triton X-100. Pretreatment of the membranes with Triton X-100 prevents the acidification. The acidification is also not observed if the [K+] and proton conductance of the membranes have been increased by the simultaneous addition of valinomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone to the K+-rich incubation medium. Either valinomycin or carbonyl cyanide p-trifluoromethoxyphenylhydrazone when added alone do not alter the response of the membranes to the addition of Na+. Na+ uptake by brush-border microvilli is enhanced in the presence of a proton gradient directed from the intravesicular space to the incubation medium. Under these conditions a transient accumulation of Na+ inside the vesicles is observed. It is concluded that intestinal and renal brush-border membranes contain a NA+/H+ antiport system which catalyses an electroneutral exchange of Na+ against protons and consequently can produce a proton gradient in the presence of a concentration difference for Na+. This system might be involved in the active proton secretion of the small intestine and the proximal tubule of the kidney.     

Sodium-chloride transport in the medullary thick ascending limb of henle's loop: Evidence for a sodium-chloride cotransport system in plasma membrane vesicles

Sodium transport mechanisms were investigated in plasma membrane vesicles prepared from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. The uptake of 22Na into the plasma membrane vesicles was investigated by a rapid filtration technique. Sodium uptake was greatest in the presence of chloride; it was reduced when chloride was replaced by nitrate, gluconate or sulfate. The stimulation of sodium uptake by chloride was seen in the presence of a chloride gradient directed into the vesicle and when the vesicles were equilibrated with NaCl, KCl plus valinomycin so that no chemical or electrical gradients existed across the vesicle (tracer exchange experiments). Furosemide decreased sodium uptake into the vesicles in a dose-dependent manner only in the presence of chloride, with a Ki of around 5 X 10(-6) M. Amiloride, at 2 mM, had no effect on the chloride-dependent sodium uptake. Similarly, potassium removal had no effect on the chloride-dependent sodium uptake and furosemide was an effective inhibitor of sodium uptake in a potassium-free medium. The results show the presence of a furosemide-sensitive sodium-chloride cotransport system in the plasma membranes of the medullary TALH. There is no evidence for a Na+/H+ exchange mechanism or a Na+ -K+ -Cl- cotransport system. The sodium-chloride cotransport system would effect the uphill transport of chloride against its electrochemical potential gradient at the luminal membrane of the cell.     

A two sodium ion/D-glucose symport mechanism: membrane potential effects on phlorizin binding

Apical membrane vesicles isolated from a continuous renal cell line, LLC-PK1, catalyze electrogenic Na+-stimulated hexose transport and Na+-dependent binding of 3H-labeled 1-[2-(beta-D-glucopyranosyloxy)-4, 6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone [( 3H]phlorizin), a competitive ligand of this transport system. Phlorizin was not itself transported across the membrane and thus can serve as a probe of the binding step. The stoichiometry of Na+-dependent phlorizin binding in vesicles was 1:1, whereas Na+/hexose cotransport in vesicles exhibited a 2:1 stoichiometry. Na+ increased the affinity of phlorizin binding without affecting the total number of binding sites. An increased number of Na+-dependent phlorizin binding sites was observed under conditions of interior-negative membrane potential. These results are consistent with a model of the Na+/glucose cotransport cycle in which the unloaded transporter is negatively charged and its orientation influenced by membrane potential. Glucose and one sodium ion interact with the transporter, resulting in an uncharged complex. Binding of a second sodium ion triggers translocation of glucose and both sodium ions via formation of a loaded carrier complex bearing a single positive charge.     

A hydrolase-related transport system is not required to explain the intestinal uptke of glucose liberated from phlorizin

The fate of [3H]glucose released from a wide range of [3H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na+-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2-2.0 mM phlorizin, the [3H]glucose uptake was a constant 11-12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [3H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [14C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [14C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na+-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medum by virtue of the position of the site where it is formed, i.e inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings.     

Inhibition of sodium for sodium exchange by phlorizin in frog sartorius muscle

The effects of phlorizin (2 X 10(-3) mol X l-1) on the Na transport of frog (Rana esculenta) sartorius muscle were investigated in glucose-free medium. Phlorizin decreased the rate coefficient of 24Na efflux by about 40%. The degree of inhibition was comparable to that caused by ouabain (10(-4) mol X l-1). Phlorizin could evoke a further reduction in the 24Na efflux also in the presence of ouabain. The intracellular Na content of the phlorizin-treated muscles remained unchanged, in contrast to a 60% increase induced by ouabain. 42K uptake was not affected by phlorizin. Data indicate that the ouabain-sensitive Na-K pump was not involved in the action of phlorizin. At the same time, phlorizin failed to alter the residual 24Na efflux measured in Li-Ringer solution containing ouabain. When Na: Na exchange was restored by replacing Na into the washout solution in the presence of ouabain, the increase of 24Na efflux was significantly diminished by phlorizin. Phlorizin reduced the 24Na uptake into a compartment with a half time of 6 min by about 40% without affecting the intracellular compartment. The results suggest that phlorizin inhibits the ouabain-insensitive Na: Na exchange in a superficial Na compartment.     

Identification of proximal tubular transport functions in the established kidney cell line, OK

OK cells, derived from an American opossum kidney, were analyzed for proximal tubular transport functions. In monolayers, L-glutamate, L-proline, L-alanine, and alpha-methyl-glucopyranoside (alpha-methyl D-glucoside) were accumulated through Na+-dependent and Na+-independent transport pathways. D-Glucose and inorganic sulfate were accumulated equally well in the presence or absence of Na+. Influx of inorganic phosphate was only observed in the presence of Na+. Na+/alpha-methyl D-glucoside uptake was preferentially inhibited by phlorizin and D-glucose uptake by cytochalasin B. An amiloride-sensitive Na+-transport was also identified. In isolated apical vesicles (enriched 8-fold in gamma-glutamyltransferase), L-glutamate, L-proline, L-alanine, alpha-methyl D-glucoside and inorganic phosphate transport were stimulated by an inwardly directed Na+-gradient as compared to an inwardly directed K+-gradient. L-Glutamate transport required additionally intravesicular K+. D-Glucose transport was similar in the presence of a Na+- and a K+-gradient. Na+/alpha-methyl D-glucoside uptake was inhibited by phlorizin whereas cytochalasin B had no effect on Na+/D-glucose transport. An amiloride-sensitive Na+/H+ exchange mechanism was also found in the apical vesicle preparation. It is concluded that the apical membrane of OK cells contains Na+-coupled transport systems for amino acids, hexoses, protons and inorganic phosphate. D-Glucose appears a poor substrate for the Na+/hexose transport system.     

Na(+)-dependent D-mannose transport at the apical membrane of rat small intestine and kidney cortex

The presence of a Na(+)/D-mannose cotransport activity in brush-border membrane vesicles (BBMV), isolated from either rat small intestine or rat kidney cortex, is examined. In the presence of an electrochemical Na(+) gradient, but not in its absence, D-mannose was transiently accumulated by the BBMV. D-Mannose uptake into the BBMV was energized by both the electrical membrane potential and the Na(+) chemical gradient. D-Mannose transport vs. external D-mannose concentration can be described by an equation that represents a superposition of a saturable component and another component that cannot be saturated up to 50 microM D-mannose. D-Mannose uptake was inhibited by D-mannose > D-glucose>phlorizin, whereas for alpha-methyl glucopyranoside the order was D-glucose=phlorizin > D-mannose. The initial rate of D-mannose uptake increased as the extravesicular Na(+) concentration increased, with a Hill coefficient of 1, suggesting that the Na(+):D-mannose cotransport stoichiometry is 1:1. It is concluded that both rat intestinal and renal apical membrane have a concentrative, saturable, electrogenic and Na(+)-dependent D-mannose transport mechanism, which is different from SGLT1.     

The 24Na-transport increasing effect of veratrine in frog sartorius muscle influenced by the tonicity, composition and temperature of the external environment.

The influence of tonicity, ionic composition and temperature of the incubating medium on the increasing effect of veratrine on 24Na transport in the frog sartorius muscle has been studied. (1) The effect of veratrine applied during 24Na loading on the rate coefficient for sodium loss depended on the tonicity of the medium. The rate of loss of 24Na from muscles loaded in the presence of veratrine was not affected if the muscles had been equilibrated in hypertonic medium. However, when treating the muscles with veratrine in isotonic medium during 24Na loading, we obtained a twofold increase in the rate coefficient for sodium loss. (2) The effect of veratrine applied during the desaturation period on 24Na efflux was also found to depend on the tonicity of the medium. Veratrine applied during the desaturation period increased the 24Na efflux in muscles equilibrated in isotonic Ringer's solution. However, when the muscles were equilibrated in hypertonic medium, veratrine did not influence 24Na efflux, not even after the rate of 24Na loss had been decreased by ouabain. (3) Hypertonic medium inhibited the Li uptake-enhancing effect of veratrine, while in isotonic medium veratrine had a marked enhancing effect. (4) In hypertonic medium lithium inhibited the otherwise characteristic increasing effect of veratrine on 24 Na uptake. (5) The increase of intracellular sodium concentration as a result of incubation in cold, potassium-free Ringer's solution did not influence the 24Na exchange-increasing effect of veratrine in isotonic medium. (6) The increasing effects of 0.1 and 0.5 mM veratrine on 24Na influx had the same degree at room temperature. However, at 5 degrees C 0.5 mM veratrine increased 24Na influx to a greater extent than 0.1 mM. (7) On the basis of our earlier experiments it has been suggested that the site of action of the 24Na uptake-increasing effect of veratrine could be the neural structures in the muscle equilibrated in hypertonic media. The present experiments confirm this suggestion and at the same time demonstrate that there are substantial differences in the mechanism of the sodium transport of veratrine-treated neural and muscle membranes, which become more apparent in hypertonic medium.     

SOME PROBLEMS INHERENT IN TRANSPORT STUDIES IN SYNAPTOSOMES

A technique utilizing a 30-place manifold has been developed to study synaptosomal transport; some problems associated with such studies have been identified and clarified. The time course of L-glutamic acid uptake has been used to test variations in experimental protocol. Synaptosomes apparently become increasingly labile with increased time of incubation. This is indicated by a drop in the curve of uptake vs time after 8–12 min. Ninety seven to 98% of the glutamate taken up from a 10^?6m solution is released by osmotic shock. Synaptosomes can be stored in 0.32 m ice-cold sucrose suspension for periods up to 50 min without decline in measured uptake. Storage for 3 h or more results in a very substantial decline in measured uptake. Neither the decline in measured uptake with time, nor the decline with storage, is prevented by increasing the osmolarity of the solutions used or by use of synaptosomes from the initial 1085 g supemate rather than after sedimentation and resuspension. Although prewarming synaptosomes at 30°C for 20 min prior to their use lessened or eliminated the decline following peak uptake, the difference between stored and non-stored synaptosomes was not improved. Uptake was also much less when synaptosomes were used from the first supernate or when warmed prior to their use. Storage of tissue prior to homogenization resulted in synaptosomes that gave minimal reductions in measured uptake. Washing synaptosomes after separation from incubation medium resulted in a variable loss of substrate radioactivity, depending on such variables as brand of filter, pore size, composition of wash solution, and temperature of wash solution. The results support the hypothesis that washing causes lysis of a portion of the synaptosomes. However, with Millipore filters (0.45 μm) and a 30°C Krebs-Henseleit wash solution, the loss caused by washing is minimized (about 15%). Measured uptake is found to depend on the type of filter used. Uptake is much greater with Millipore 0.45 pm filters than with Gelman 0.45 μm filters. Use of Nuclepore (0.4 μm) filters results in measured uptakes only about 5% of that when Millipore 0.45 μm filters are used. With Millipore filters, 0.30 μm pore size filters gave uptakes only 68% of that using 0.45 pm pore size filters.     

Monoclonal antibodies that bind the renal Na+/glucose symport system. 1. Identification

Phlorizin is a specific, high-affinity ligand that binds the active site of the Na+/glucose symporter by a Na+-dependent mechanism but is not itself transported across the membrane. We have isolated a panel of monoclonal antibodies that influence high-affinity, Na+-dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK1, a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG2b, reproducibly stimulated Na+-dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na+-dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, taken together with results in the following paper [Wu, J.-S.R., & Lever, J.E. (1987) Biochemistry (following paper in this issue)], provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na+/glucose symporter.     

Renal sodium transport in vitamin D resistant hypophosphatemic rickets

To investigate the possible role of a Na transport defect in the pathogenesis of the phosphaturia in vitamin D resistant rickets, we studied the activity of the Na-K ATPase activity along the microdissected segments of the nephron in normal (N) and hypophosphatemic mice (Hyp), the Na uptake by renal brush border membrane (BBM), as well as the interrelationship between Na and phosphate transport through this membrane. In N mice, Na-K ATPase activity was present in decreasing order, in the distal tubule, the ascending branch of the loop of Henle, the proximal tubule, and the collecting tubule. In Hyp mice, the Na-K ATPase activity was comparable to that measured in N mice, except in the granular segment of the distal tubule where a 256% of the control activity was reproducibly observed. In N mice, Na initial uptake by BBM vesicles increased with Na concentration in the incubation medium, according to two kinetic components: one saturable, evident at low substrate concentrations and the other, nonsaturable, corresponding to a passive diffusion. The addition of 5 mM PO4 in the incubation medium did not significantly influence Na transport. In contrast, Na concentration in the incubation medium largely modified the kinetics of PO4 uptake: increasing Na concentration enhanced PO4 uptake and decreased the apparent Km. In Hyp mice, Na uptake by BBM was identical to that observed in N mice, but PO4 uptake was decreased by half. Na concentration in the incubation medium similarly influenced PO4 uptake in N and Hyp mice, and the Km values at each concentration of Na were comparable in the two series of animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characteristics of uptake of short chain fatty acids by luminal membrane vesicles from rabbit kidney

The mechanisms of renal transport of short chain fatty acids by luminal membrane vesicles prepared from pars convoluta or pars recta of rabbit proximal tubule were studied by a Millipore filtration technique and by a spectrophotometric method using a potential-sensitive carbocyanine dye. Both luminal membrane vesicle preparations take up propionate and butyrate by strictly Na+-dependent transport systems, although with different characteristics. The uptake of short chain fatty acids by membrane vesicles from the pars convoluta was insensitive to changes in membrane potential, which is indicative of electroneutral transport of these compounds. Furthermore, kinetic studies showed that the Na+-dependent, but electrically silent transport of propionate is saturable (Km = 10.9 +/- 1.1 mM and Vmax = 3.6 +/- 0.2 nmol/mg protein per 20 s) and is unaffected by the presence of L- and D-lactate, indicating that these monocarboxylic acids did not share the same common transport system. In the luminal membrane vesicles from the pars recta, the uptake of propionate and butyrate was mediated by an Na+-dependent electrogenic transport process, since addition of the organic compounds to these vesicle/dye suspensions depolarized the membrane vesicles and the renal uptake of propionate and butyrate was enhanced by K+ diffusion potential induced by valinomycin. Competition experiments revealed that in contrast to the transport of propionate by vesicles from the pars convoluta, the Na+-dependent electrogenic transport of short chain fatty acids in vesicles from the pars recta occurred via the same transport system that is responsible for the reabsorption of L- and D-lactate in this region of rabbit kidney proximal tubule.     

Enhanced glucose absorption in the rat small intestine following repeated doses of 5-fluorouracil

Many studies demonstrated that 5-fluorouracil (5-FU) treatment of rodents caused the damage of small intestine, resulting in the malabsorption, while we recently found that repeated administration of 5-FU to rats increased Na(+)-dependent glucose absorption in the small intestine. This study investigated the cause of enhanced glucose absorption. 3-O-methyl-d-glucose (3-OMG) absorption was examined using the everted intestine technique. d-Glucose uptake, phlorizin binding, Western blot analysis and membrane fluidity were examined using small intestinal brush-border membrane vesicles (BBMV). Repeated oral administration of 5-FU to rats increased Na(+)-dependent 3-OMG absorption in the small intestine, while alkaline phosphatase activity in the small intestine decreased. Na(+)/K(+)-ATPase activity of 5-FU-treated rats was about three-fold higher than that of control rats. Although the amount of Na(+)-dependent glucose co-transporter (SGLT1) in 5-FU-treated rats decreased, the overshoot magnitude of d-glucose uptake in BBMV was not altered. Maximum binding of phlorizin in 5-FU-treated rats was 1.5-fold larger than that of control rats, but not altered the maximal rate of d-glucose absorption, Michaelis constant of d-glucose and dissociation constant of phlorizin. The membrane fluidity of 5-FU-treated rats increased. The enhanced d-glucose absorption in 5-FU-treated rats seems to occur secondarily due to the activation of Na(+)/K(+)-ATPase activity in basolateral membranes (BLM). Because the amounts of SGLT1 in 5-FU-treated rats decreased, the increase of turnover rate of SGLT1 and/or an expression of unknown Na(+)-dependent glucose co-transporter with high affinity for d-glucose and phlorizin sensitivity would contribute to the enhancement of d-glucose transport in 5-FU-treated rats.