Rat Brush Border Membrane Vesicles Research Articles

Brush Border Membrane Vesicles from Rats and Gerbils Can Be Utilized to Evaluate the Intestinal Uptake of All-trans and 9-cis β-Carotene

Intestinal brush border membrane vesicles (BBMV) have previously been used to investigate the absorptive processes of a wide variety of nutrients. The first objective of this work was to develop a BBMV system to study carotenoid uptake using rat small intestine. The uptake of all-trans β-carotene by rat BBMV was linear up to 5.9 µmol/L, suggesting passive uptake at the concentrations used in this study. Both all-trans β-carotene and 9-cis β-carotene were taken up at a similar rate by the rat BBMV. The vesicle system was then utilized to investigate the uptake of retinol and β-carotene by BBMV isolated from Mongolian gerbils which had been subjected to three different vitamin A depletion and refeeding protocols. Three groups of gerbils (n = 10/group) were fed vitamin A-deficient diets for 8 wk. One group was killed at that point (marginally depleted), a second group was refed vitamin A for 5 d and a third group was further depleted of vitamin A for 5 d. Pooled BBMV from the marginally depleted group demonstrated greater uptake of all-trans β-carotene than pooled BBMV from the other two groups of gerbils while those from the group refed vitamin A for 5 d had greater uptake than the group that was depleted for 5 additional days (P < 0.01). In contrast, the uptake of retinol by BBMV was not significantly different among the three groups of gerbils. The results of the gerbil study suggest that as they become increasingly vitamin A deficient, impairment of all-trans β-carotene uptake by intestinal mucosal cells may be taking place. The BBMV system should prove valuable for the evaluation of the intestinal mucosal cell membrane uptake of carotenoids and retinoids under various nutritional conditions.

Inhibitory effects of extractives from leaves of Morus alba on human and rat small intestinal disaccharidase activity

The inhibitory effect on human and rat intestinal disaccharidase by the extractive from the leaves of Morus alba (ELM) containing 0.24 % 1-deoxynojirimycin equivalent and its inhibitory activities were investigated by the modified Dahlqvist method. In the presence of 1000-fold diluted ELM solution, the sucrase activity of four human samples was inhibited by 96 % and that of maltase and isomaltase by 95 and 99 %, respectively. The activities of trehalase and lactase were inhibited by 44 and 38 %, respectively. The human disaccharidase activities varied from sample to sample because the samples were obtained from different resected regions after surgery. However, the ratio of the inhibitory effect for sucrase, maltase, isomaltase, trehalase and lactase was very similar among the four samples, and also that of resembled rat intestinal disaccharides. The inhibitory constant of the 1-deoxynojirimycin equivalent for sucrase, maltase and isomaltase was 2.1 x 10(-4), 2.5 x 10(-4) and 4.5 x 10(-4) mm, respectively, and these inhibitory activities were shown, using rat brush border membrane vesicles, to be competitive. These results demonstrate that digestion is inhibited when an appropriate amount of ELM is orally ingested with sucrose or polysaccharide in man. When ELM was orally administered in a sucrose solution to fasted rats, the elevation in blood glucose was significantly suppressed, depending on the concentration of ELM given. These results suggest that ELM could be used as an ingredient in health foods and in foods that help to prevent diabetes.

O0092 PSYCHOLOGICAL STRESS ALTERS GLUCOSE ABSORPTION IN RAT JEJUNUM

Introduction: Humans frequently react to chronic stress with decreased appetite and loss of body weight. In our experiments, we have repeatedly observed that rats exposed to chronic psychological stress fail to gain weight in spite of normal food intake. We hypothesized that nutrient absorption may be impaired by psychological stress, explaining the reduced weight gain. This study was designed to investigate glucose absorption in rats submitted to different periods of water avoidance stress (WAS). Methods: Rats were subjected to WAS (1 h/day) for 1, 5 or 10 consecutives days. Body weight and food intake were recorded daily before and during the stress period. Four hours after the last WAS session, rats were sacrificed and segments of jejunum mounted in Ussing chambers to study the flux of glucose (using 3H-3-o-methylglucose) and SGLT-1 kinetics. Mucosa was obtained to prepare brush border membrane (BBM) vesicles, used to measure 14C-fructose uptake as well as SGLT-1 and GLUT2 expression by Western blots. Results: Net absorptive flux (Jnet) of glucose decreased with the duration of the stress procedure, due to both a decrease in Jm-s and an increase in Js-m (Table). The decrease in Jm-s was paralleled by a decrease of Vmax of SGLT-1. The increase in Js-m could not be accounted for by an increase in paracellular permeability since the conductance (G) was not altered by stress. On the other hand, we observed a time-dependent increase in phloretin-sensitive uptake of 14C-fructose by BBM vesicles of rats after 1, 5 or 10 days of WAS, which was not present in control animals. This suggested the presence of GLUT2, a transporter able to transport glucose or fructose in both directions, in the BBM. Western blots confirmed the increase in GLUT2 expression in the BBM after 1, 5 and 10 days of WAS.Table 1Conclusion: Psychological stress induces a decrease in the absorption of glucose in the jejunum of rats resulting from a concomitant decrease in SGLT-1 activity and an increase in GLUT2 expression in the enterocyte apical membrane. Glucose malab-sorption may explain the growth arrest observed in rats under stress. In addition, altered mucosal metabolism may be involved in the stress-induced changes in mucosal barrier function.

A phosphorylated phloretin derivative. Synthesis and effect on intestinal Na(+)-dependent phosphate absorption.

2'-Phosphophloretin (2'-PP), a phosphorylated derivative of the plant chalcone, was synthesized. The effect of 2'-PP, on Na(+)-dependent phosphate uptake into intestinal brush-border membrane vesicles (BBMV) isolated from rabbit and rat duodenum and jejunum was examined. 2'-PP decreased Na(+)-dependent phosphate uptake into rabbit BBMV with an IC(50) of 55 nM and into rat BBMV with an IC(50) of 58 nM. 2'-PP did not affect Na(+)-dependent glucose, Na(+)-dependent sulfate, or Na(+)-dependent alanine uptake by rabbit intestinal BBMVs. 2'-PP inhibition of rabbit intestinal BBMV Na(+)-dependent phosphate uptake was sensitive to external phosphate concentration, suggesting that 2'-PP inhibition of Na(+)-dependent phosphate uptake was competitive with respect to phosphate. Binding of [(3)H]2'-PP to rabbit intestinal BBMV was examined. Binding of [(3)H]2'-PP was Na(+)-dependent with a K(0.5) for Na(+)(Na(+) concentration for 50% 2'-PP binding) of 30 mM. The apparent K(s) for Na(+)-dependent [(3)H]2'-PP binding to rabbit BBMVs was 58 nM in agreement with the IC(50) for 2'-PP inhibition of Na(+)-dependent phosphate uptake. These results indicate that 2'-PP bound to rabbit or rat intestinal BBMV Na(+)-phosphate cotransporter and inhibited Na(+)-dependent phosphate uptake. In rats treated with 2'-PP by daily gavage, the effect of 2'-PP on serum phosphate, serum glucose, and serum calcium was examined. In a concentration-dependent manner, 2'-PP reduced serum phosphate by 45% 1 wk after starting treatment. 2'-PP did not alter serum calcium or serum glucose. The apparent IC(50) for 2'-PP in vivo was 3 microM.

Significance of substrate hydrophobicity for recognition by an oligopeptide transporter (PEPT1).

Our previous paper [(1999) Bioconjugate Chem. 10, 24-31] pointed out that hydrophobicity of substrates/inhibitors plays an important role in the recognition by an oligopeptide transporter (PEPT1) expressed in the human intestinal epithelial cell line Caco-2. To determine the significance of that hydrophobicity, we have now synthesized dipeptide analogues conjugating the epsilon-amino group of Lys in Val-Lys with aliphatic carboxylic acids: acetic acid (C2), propanoic acid (C3), pentanoic acid (C5), hexanoic acid (C6), and decanoic acid (C10). The affinities of these conjugates were estimated by their inhibition of the accumulation rate of Gly-Sar, a well-established substrate for PEPT1. With the increase in length of the hydrocarbon chain of the conjugates, i.e., in the hydrophobicity of the conjugates, the inhibition strengthened. Dixon-Webb plot analysis of the inhibition by the C10-conjugated dipeptide showed competitive inhibition. The trans-stimulation effect of Val-Lys conjugated to C10 or C5 on the uptake of Ceftibuten was observed using rat brush border membrane vesicles. This findings showed that these conjugates are transportable substrates. These results confirmed that the hydrophobicity of substrates/inhibitor is one of the factors in the recognition by PEPT1.

Inhibition of intestinal absorption of 5-methyltetrahydrofolate by fluoxetine

Thein vitro uptake of 5-methyltetrahydrofolate (5-MeTHF) by rat and human intestine is dose-dependently inhibited by the antidepressant drug fluoxetine (FLX). In rat jejunum rings, 0.2 mM FLX inhibited the uptake of 5-MeTHF (0.25 μM) by 32% (15 min) and 49% (45 min). In brush border membrane vesicles (BBMV) from rat jejunum, 0.2 mM FLX inhibited the folate uptake at the overshoot (90 s) by 40 %. Similar inhibition was observed with human Caco-2 cells and duodenal biopsies. FLX action is exerted on the active transport component of the folate uptake, since the drug has no effect when the passive diffusion component becomes prominent by high substrate concentration, or by 0-4 oC incubation or by addition of the folate transport inhibitor DIDS (1mM). The kinetic analysis with rat BBMV suggests a non-competitive inhibition of the 5-MeTHF transport by FLX, with apparent values for KM = 0.89 μM, Vmax = 1.89 pmol/mg prot./10 s, and KI = 0.21 mM. After 21 days of treatment with FLX (10 mg/kg/day), the folate uptake by jejunum rings or by BBMV from the treated rats was diminished, and the folate levels in erythrocytes and serum were also decreased.

IFN-gamma downregulates expression of Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and human Caco-2/bbe cells.

Diarrhea associated with inflammatory bowel diseases has traditionally been attributed to stimulated secretion. The purpose of this study was to determine whether chronic stimulation of intestinal mucosa by interferon-gamma (IFN-gamma) affects expression and function of the apical membrane Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and Caco-2/bbe (C2) cells. Confluent C2 cells expressing NHE2 and NHE3 were treated with IFN-gamma for 2, 24, and 48 h. Adult rats were injected with IFN-gamma intraperitoneally for 12 and 48 h. NHE2 and NHE3 activities were measured by unidirectional (22)Na influx across C2 cells and in rat brush-border membrane vesicles. NHE protein and mRNA were assessed by Western and Northern blotting. IFN-gamma treatment of C2 monolayers caused a >50% reduction in NHE2 and NHE3 activities and protein expression. In rats, region-specific, time- and dose-dependent reductions of NHE2 and NHE3 activities, protein expression, and mRNA were observed after exposure to IFN-gamma. Chronic exposure of intestinal epithelial cells to IFN-gamma results in selective downregulation of NHE2 and NHE3 expression and activity, a potential cause of inflammation-associated diarrhea.

Ontogeny of Na+/H+ antiporter activity in rat proximal convoluted tubules.

Neonates have a lower serum bicarbonate level than adults, which is caused by a lower renal threshold for bicarbonate. Eighty percent of bicarbonate reabsorption occurs in the proximal tubule, in which proton secretion is predominantly mediated by a luminal Na+/H+ antiporter. Previous studies have demonstrated that there is a maturational increase in apical membrane rabbit proximal convoluted tubule Na+/H+ antiporter activity. However, in rat brush border membrane vesicles, Na+/H+ activity was higher in neonates than that in adult rats. To examine the maturation of Na+/H+ antiporter activity in rat proximal convoluted tubules, we perfused rat proximal convoluted tubules in vitro. Na+/H+ antiporter activity was assayed as the proton secretory rate on luminal sodium removal. Na+/H+ antiporter activity was 121.2 +/- 18.4 pmol/mm x min in neonatal and 451.8 +/- 40.6 pmol/mm x min in adult proximal convoluted tubules (p < 0.001). We next examined whether the increase in Na+/H+ antiporter activity was associated with changes in renal cortical NHE3 mRNA and brush border membrane NHE3 protein abundance. Adult renal cortical NHE3 mRNA abundance was 10-fold greater than that in 1-d-old neonates (p < 0.001). There was a comparable developmental increase in renal brush border membrane vesicle NHE3 protein abundance (p < 0.001). In summary, this study demonstrates that there is a maturational increase in rat apical membrane Na+/H+ antiporter activity, renal cortical NHE3 mRNA, and brush border membrane vesicle NHE3 protein abundance.

Renal excretion mechanism of NS-49, a phenethylamine class alpha 1A-adrenoceptor agonist.

Renal handling of NS-49, which is an organic cation and a chiral compound, was investigated in rats, rabbits and dogs. Renal clearance (Cl(re)) of NS-49 was 3.4-fold the glomerular filtration rate (GFR) in the rat in vivo study. The clearance ratio (Cl(re)/GFR) approached unity during cimetidine infusion. Change in the urine flow rate or urinary pH did not affect the Cl(re) of NS-49. The stop-flow patterns of NS-49 in the rabbits and dogs showed a secretion peak in the proximal tubules. On concomitant administration of cimetidine, the secretion peak disappeared, the stop-flow pattern showing neither a secretion nor reabsorption peak. These findings indicate that in these species NS-49 undergoes glomerular filtration and extensive proximal tubular secretion, but little reabsorption. A transport mechanism study of NS-49 in brush-border membrane vesicles (BBMVs) isolated from rat kidney cortex showed that it is transported via the carrier-mediated H(+)/organic cation antiport system. In the rat renal clearance studies (in vivo) tubular secretion of NS-49 was significantly inhibited by quinine (p<0.01) but not by quinidine. Transport studies done with rat BBMVs (in vitro) also showed quinine to be more potent than quinidine in inhibiting NS-49 uptake. These results indicate that stereoselective interaction occurs in active renal tubular secretion.

Effects of zinc and copper on cadmium uptake by brush border membrane vesicles

The effects of essential metals, zinc (Zn) and copper (Cu), on cadmium (Cd) uptake were investigated in brush border membrane vesicles (BBMV) isolated from the rat renal cortex and LLC-PK 1 cells. BBMV were incubated with Cd in the presence or absence of Zn or Cu, and then washed with a chelating agent, EGTA, to remove Cd bound to the outer surface of BBMV. Co-incubation with Zn or Cu decreased Cd accumulation in these BBMV in a concentration-dependent manner. Kinetic analysis of the initial accumulation of Cd suggested that Cd is taken up into rat BBMV via an unsaturable component and a saturable component ( K m = 13.8 μM, V max = 1.44 nmol/mg protein/min), and co-incubation with Zn significantly increased the K m of the saturable component without affecting the V max, whereas Cu significantly increased the K m-value and decreased the V max-value. Increasing the osmolarity of the incubation medium slightly decreased Cd accumulation in the absence of Zn or Cu, whereas it did not decrease Cd accumulation in the presence of these metals. These results suggest the possibility that, in addition to passive diffusion, Cd is also taken up from the renal brush border membrane via carrier-mediated mechanisms that are inhibited by Zn competitively and by Cu non-competitively. Furthermore, these results suggest that: (1) Cd binds externally and internally to BBMV, (2) little Cd is transported into the intravesicular space, and (3) both Zn and Cu decrease the binding and transport of Cd.

37] Measurements and characteristics of intestinal riboflavin transport

Publisher Summary The transport of labeled riboflavin (RF) in intestinal intact tissue and in cellular preparations from differing animal species, at low substrate concentrations, was found to involve a carrier-mediated process that could be inhibited by metabolic inhibitors, by unlabeled RF, and by some of its structural analogs. Intestinal absorption includes transport of RF across both enterocyte membranes (brush border and basolateral membranes) and cytoplasm, where RF can be enzymatically esterified. Each of these steps can be examined separately by means of appropriate intestinal preparations. This chapter describes certain characteristics of RF transport both in rat brush border membrane vesicles, which enable investigation into the pure membrane process of cellular entry, and in isolated enterocytes, which allow intracellular RF metabolism to be investigated. This chapter also describes a high-performance liquid chromatography (HPLC) method that is suitable for an investigation of RF intracellular biotransformation.

Mucoadhesive polymers in peroral peptide drug delivery. V. Effect of poly(acrylates) on the enzymatic degradation of peptide drugs by intestinal brush border membrane vesicles

The purpose of the study was to evaluate the inhibitory effect of the mucoadhesive poly(acrylates) polycarbophil and carbomer on the activity of proteolytic enzymes bound to the intestinal brush border. To that end, the degradation of a number of peptide drugs in the presence or absence of the poly(acrylates) was investigated, using rat brush border membrane vesicles (BBMV) as the protease preparation. Both carbomer and polycarbophil in concentrations of 0.25 and 0.5% (w/v) reduced rather weakly the enzymatic degradation of the peptide 9-desglycinamide, 8-arginine vasopressin (DGAVP), and only 0.5% (w/v) carbomer inhibited metkephamid degradation, but not polycarbophil. More pronounced inhibitory effects on DGAVP breakdown were found following a 30 min preincubation of the BBMV suspension with 0.5% (w/v) carbomer. However, the poly(acrylic acid) derivatives were unable to inhibit the degradation of buserelin at all. On the other hand, the polypeptide hormone insulin was remarkably stable in the BBMV preparations. In conclusion, the poly(acrylic acid) derivatives polycarbophil and carbomer show rather weak inhibitory effects on enzymes of the intestinal brush border cell membranes responsible for DGAVP and metkephamid degradation.

Oral Absorption of Anti-Acquired Immune Deficiency Syndrome Nucleoside Analogues. 2. Carrier-Mediated Intestinal Transport of Stavudine in Rat and Rabbit Preparations

AbstractThe intestinal transport and metabolism of stavudine (d4T), a nucleoside analogue of thymidine used in the treatment of AIDS, was studied using single-pass intestinal perfusion (SPIP), intestinal brush-border membrane vesicles (BBMV), and mucosal homogenates in rats and rabbits. In the SPIP, d4T demonstrated concentration-dependent mean wall permeability (P *w) at perfusate concentrations ranging from 0.001 to 25mM. In coperfusion studies using 0.1mM thymidine, 1mM formycin B, or 5 μM NBTI as putative inhibitors of d4T transport, the P *w of 5 μM d4T was reduced to 48%, 62%, and 70% of the control value, respectively, suggesting the involvement of multiple nucleoside carriers in the intestinal uptake of d4T. d4T uptake in rat BBMV was significantly greater in the presence of a sodium ion gradient compared with a sodium-free (choline) gradient. The permeability of d4T, in the presence of a sodium gradient, was concentration-dependent and inhibited by 10mM thymidine but not significantly reduced by 10mM formycin B. In the presence of 10 μM NBTI, the permeability of d4T was not inhibited; however, the binding of d4T to rat and rabbit BBMV was significantly reduced. Formycin B did not significantly reduce the d4T uptake in rat or rabbit BBMV suggesting that d4T does not interact with the purine-selective N1 nucleoside carrier. However, because formycin B inhibited d4T uptake in the SPIP and since d4T inhibited formycin B uptake in rat but not rabbit BBMV, it appears to interact with the N3 carrier which has been demonstrated in rat but not rabbit intestine. Also, an interaction with the sodium-independent facilitative transporter at the basolateral membrane cannot be ruled out. The low hybrid Km and high passive permeability of d4T likely account for the lack of saturable absorption behavior observed in humans, whereas the brush-border and intracellular stability of d4T preserve the high bioavailability observed after oral dosing.

Ferrous iron uptake by rat duodenal brush border membrane vesicles: Effects of dietary iron level and competing minerals (Zn +2, Mn +2, and Ca +2)

We sought to confirm a recent report that Fe +2 uptake into rat brush-border membrane vesicles is markedly increased by short-term consumption of iron-deficient diet, with no additional enhancement as the animal becomes functionally iron-deficient with continuing dietary Fe deprivation. In addition, we investigated whether previously observed in vivo absorption interactions of iron, zinc, and manganese occur in the brush border membrane vesicles uptake process, and whether short-term or long-term consumption of an iron-deficient diet affects the interaction at the uptake level. We did not observe any differences in Fe +2 uptake between normal and iron-deficient brush border membrane vesicles, even when the iron status contrast was intensified by feeding a high iron versus iron-deficient diet for 3 weeks. Equimolar Zn +2 and Mn +2 decreased Fe +2 uptake by 29 to 50% and 11 to 39%, respectively. Iron deficiency did not alter these effects. Equimolar Fe +2 decreased Zn +2 uptake by 13 to 22%. Calcium, included as a negative control, did not affect Fe +2 uptake. Thus, some competition between Fe +2 and similar divalent cations does occur at the level of the brush border membrane; the exact nature of this competition remains to be determined.

Expression of taurine transporter and its regulation by diet in Xenopus laevis oocytes following injection of rat kidney cortex mRNA.

NaCl-dependent taurine transport adapts to changes in the dietary intake of sulfur amino acids. The renal adaptive response is expressed by enhanced NaCl-dependent taurine cotransport by brush border membrane vesicles after a low taurine diet and reduced transport after a high taurine diet as compared to a normal taurine diet. In order to determine if this adaptive regulation is dependent on new protein synthesis, the Xenopus laevis oocyte expression system was utilized to define the translational regulation of taurine transporter activity. Poly(A)+ RNA was isolated from kidney cortex of Sprague Dawley rats fed either a low, normal or high taurine diet for 28 days. Injection of poly(A)+ RNA resulted in a time- and dose-dependent increase in NaCl-taurine co-transport. Taurine uptake was stimulated about 2-10-fold after injection of poly(A)+ RNA (10-40 ng) as compared to H2O-injected oocytes. Taurine uptake by oocytes was sodium- and anion-dependent (Cl- > Br- > SCN- > I-). The Km and Vmax of the taurine transporter were 22.5 microM and 8.35 pmol/h/oocyte respectively, similar to the Km of 17.0 microM found in rat brush border membrane vesicles. Because the adaptive response involves an augmented or reduced Vmax of the transporter, taurine uptake by oocytes injected with poly(A)+ RNA from rats fed each diet was examined. Poly(A)+ RNA from rats fed a low taurine diet elicited twice the taurine uptake elicited from rats fed a normal taurine diet and more than three times the uptake from high taurine-fed rats. Northern blot analysis after hybridization with an RNA probe for the taurine transporter cDNA from MDCK cells (obtained from Dr. Uchida) indicated that the molecular size of taurine transporter mRNA is about 1.9 kb and is regulated by diet. Expression of taurine transporter by the oocytes injected with 30 ng of capped transcript from pNCT was significantly reduced by taurine in the medium. In conclusion, taurine uptake by oocytes after injection of mRNA is similar to brush border membrane vesicles taurine transport. The long-term adaptive response is regulated at the level of mRNA, and the short-term adaptive response is regulated at the level of protein synthesis or secretion. We speculate that the renal adaptive response to altered dietary sulfur amino acid intake is both transcriptionally and translationally regulated.

Mechanisms of intestinal absorption of the antibiotic, fosfomycin, in brush-border membrane vesicles in rabbits and humans.

In order to clarify the mechanism of intestinal absorption of an antibiotic, fosfomycin (FOM), the uptakes of FOM by rabbit and human small intestinal brush-border membrane vesicles (BBMV) were studied. The initial uptake of FOM by BBMV at 15 s was saturable at a higher concentration of FOM. The kinetic parameters at 37 degrees C of the saturable uptake expressed by the Michaelis-Menten equation were Kt = 5.17 mM and Jmax = 3.88 nmol/15 s/mg protein for rabbits, and Kt = 4.03 mM and Jmax = 1.90 nmol/15 s/mg protein for humans. The most efficient uptake was observed in the presence of both inward-directed Na(+)- and H(+)-gradients in both mammals. The uptake of FOM was inhibited by inorganic phosphate, FOM glycol which is a degradation product of FOM in the gastric juice and specific inhibitors of phosphate transport such as arsenate and phosphonoacetic acid. These findings confirmed that FOM absorption from rabbit and human small intestines is associated with the phosphate transport system. These transport phenomena of FOM are in close agreement with those obtained previously in rat BBMV studies. Judging from the results obtained for three mammalian species, rat, rabbit and human, it was concluded that carrier-mediated transport via the phosphate transport system is a very important pathway of intestinal absorption of FOM.

Inhibition of sodium-phosphate cotransport in renal brush-border membranes with the stilbenedisulfonate, H 2-DIDS

Membrane proteins involved with sodium/phosphate cotransport across the renal brush border provide the sensitive control for phosphate homeostasis. The present study describes the inhibition of sodium/phosphate cotransport with the stilbenedisulfonate derivatives, DIDS and H 2-DIDS. Preincubation of the rat brush-border membrane vesicles with H 2-DIDS led to the inhibition of sodium-dependent phosphate uptake with a half maximal concentration, IC 50, of about 10 μM. The inhibition was irreversible supporting the notion that H 2-DIDS forms covalent bonds with the cotransporter. The cotransporter could be protected by excess sodium phosphate but not sodium chloride, sodium sulfate, sodium succinate, sodium bicarbonate, nor sodium phosphonoformate. These observations suggest that the stilbenedisulfonates may be useful in labeling the sodium/phosphate cotransporter within renal brush-border membranes.

The influence of zinc, magnesium, and iron on calcium uptake in brush border membrane vesicles.

The effects of adding increasing concentrations of magnesium (Mg), zinc (Zn), and iron (Fe) on calcium (Ca) uptake into rat brush border membrane vesicles was measured. At ratios of 1:1, based on the RDA, none of these minerals were found to significantly decrease calcium uptake. Mg, however, inhibited Ca uptake at an RDA ratio of 3:1 (Mg:Ca). At higher RDA ratios (Zn:Ca 10:1), Zn also decreased Ca uptake. In a comparison of the molar ratios of the above cations, Zn inhibited Ca uptake the most (0.12:1), possibly by competing directly at high-affinity Ca binding sites. Fe did not decrease Ca uptake but rather demonstrated an enhancing effect at high concentrations (20:1). When mixtures of the above divalent cations were studied, the effects on Ca uptake could largely be explained by the actions of the ions individually.

The effects of different membrane isolation and purification techniques on D-glucose transport into rat brush-border membrane vesicles.

Current and widely used methods for the isolation and purification of brush-border membranes involve the aggregation of non-brush-border membranes with the divalent cations Ca2+ or Mg2+ with or without subsequent exposure to chaotropic agents (e.g., KSCN). Evidence suggests that these techniques yield morphologically distinct and heterogeneous populations of membranes and that functional differences exist between membrane vesicles prepared by the different procedures, presumably reflecting this heterogeneity. To investigate the effect of the various isolation techniques on the kinetic parameters of D-glucose transport, rat intestinal brush-border vesicles were prepared by each of the following four methods: (i) Ca2+ precipitation; (ii) Ca2+ precipitation with KSCN treatment; (iii) Mg2+ precipitation; and (iv) Mg2+ precipitation with KSCN treatment. Membrane purity as indicated by the enrichment of the enzyme membrane markers sucrase and alkaline phosphatase did not differ between isolation procedures. The Mg-Na-K ATPase activity showed an enrichment factor of less than 1.0 for each of the isolation techniques. D-Glucose uptake was measured with a rapid filtration method under conditions of a zero-trans, 100 mM cis-NaSCN gradient. The membrane preparations yielded similar Hofstee transformations displaying the curvilinear relationship thought to be consistent with the existence of multiple transporters for D-glucose. The average kinetic parameters calculated from the Hofstee plots for each technique were similar. It was concluded that D-glucose transport into rat jejunal membrane vesicles was unaffected by the variation in morphology arising from the technique used to purify the membranes.

Developmental maturation of calcium transport by rat brush border membrane vesicles.

The developmental aspects of calcium transport across the intestinal brush membrane vesicles was studied utilizing a millipore filtration technique. Calcium transport represented uptake into the intravesicular space as evidenced by osmolality studies, calcium release by the calcium ionophore A23187, and temperature dependency. Calcium transport in both suckling and adolescent rats appears to occur by a saturable mechanism. Calcium uptake was similar in the presence of sodium and potassium gradients, but decreased in the presence of choline gradient. The imposition of negative membrane potential did not enhance calcium uptake compared to voltage clamp conditions indicating an electroneutral process. The initial rate of calcium uptake was linear up to 15 s. Kinetic analysis of calcium uptake at 7 s showed lower Vmax and lower Km in suckling rats compared to adolescent rats. These studies are the first to demonstrate the maturational aspects of calcium entry at the brush border level and are consistent with our previous kinetic studies utilizing whole tissue.