Isolated Brush Border Membrane Research Articles

Sphingomyelin and cholesterol modulate sodium coupled uptakes in proximal tubular cells

Sphingomyelin (SM) and cholesterol are major lipid species of apical membranes in renal proximal tubular cells and confer to these membranes a low fluidity. Changes in membrane fluidity and/or lipidic composition were shown to affect the activity of cotransport systems of renal apical membranes. We evaluated the effect of decreasing membrane SM content on lipidic composition, membrane fluidity and sodium (Na)coupled uptakes in rabbit proximal tubular cells in primary culture. Sphingomyelinase (SMase) (30 to 250 mU/ml) decreased [3H]choline-labeled SM content, decreased cholesterol content, and increased cholesterol esterification. SMase did not modify membrane fluidity on isolated brush border membranes. SMase decreased Vmax of Na-dependent uptake of phosphate and alpha-methyl-D-glucoside, but not of alanine. SMase did not influence protein kinase C-induced inhibition of phosphate and glucose uptake. Increasing membrane cholesterol content with cholesterol-enriched liposomes subsequently to SMase action restored in part glucose uptake, but not phosphate uptake. In conclusion, SM degradation affected Na-phosphate and Na-glucose cotransports through changes in both SM and cholesterol contents of apical proximal membranes; these changes seemed to occur independently from changes in bulk membrane fluidity. These results suggest that SM and cholesterol have distinct and intricated roles in accessibility and/or activity of apical cotransport systems.

High-affinity phlorizin binding in Mytilus gill

The gill of the marine mussel, Mytilus, contains a high affinity, Na-dependent d-glucose transporter capable of accumulating glucose directly from sea water. We examined the ability of the β-glucoside, phlorizin, to act as a high-affinity ligand of this process in intact gills and isolated brush border membrane vesicles (BBMV). The time course of association of nanomolar [ 3H]phlorizin to gills and BBMV was slow, with t 50 values between 10 and 30 min, and a half-time for dissociation of approx. 30 min. 1 mM d-glucose reduced equilibrium binding of 1 nM phlorizin by 90–95%, indicating that there was little non-specific binding of this ligand to the gill. In addition, there was little, if any hydrolysis by the gill of phlorizin to its constituents, glucose and phloretin. Phlorizin binding to gills and BBMV was significantly inhibited by the addition of 50 μM concentrations of d-glucose and α-methyl- d-glucose, and unaffected by the addition of l-glucose and fructose. Binding to gills and BBMV was reduced by > 90% when Na + was replaced by K +. Replacement of Na + by Li + effectively blocked binding to the intact gill, although Li + did support a limited amount of glucose-specific phlorizin binding in BBMV. The K d values for glucose-specific phlorizin binding in intact gills and BBMV were 0.5 nM and 6nM, respectively. We conclude that phlorizin binds with extremely high affinity to the Na-dependent glucose transporter of Mytilus gill, which may be useful in future efforts to isolate and purify the protein(s) involved in integumental glucose transport.

The influence of mannitol on myoglobinuric acute renal failure

This study was undertaken to explore the protective influence of mannitol against the glycerol model of myohemoglobinuric acute renal failure. Three hypotheses were tested: (1) mannitol confers cytoprotection by acutely blunting renal hypoperfusion, thereby improving tubular cell energetics; (2) as an hydroxyl radical (OH.) scavenger, mannitol mitigates Fe-driven lipid peroxidation and, hence, decreases tubular cell necrosis; and (3) mannitol prevents intrarenal heme pigment trapping, decreasing cast formation. Rats were injected with 50% glycerol (10 mL/kg im), followed immediately by an iv mannitol (1.25 mL/100 g over 1 h) or sham infusion. Mannitol induced a brisk diuresis (approximately 5.7 mL/2 h; approximately 35 mg of heme protein excreted), whereas glycerol controls were anuric. Mannitol did not significantly increase postglycerol RBF (2.8 mL/min), and it paradoxically worsened cellular energetics, halving cortical ATP concentrations at 1 h. However, this adverse effect on ATP was transient, correlating with active diuresis. Glycerol did not induce convincing in vivo lipid peroxidation (malondialdehyde; conjugated diene assay), and mannitol did not block Fe-driven in vitro lipid peroxidation of isolated brush border membrane vesicles. Na benzoate, an OH. scavenger, conferred no in vivo or in vitro protection. However, Na2SO4, not an OH. scavenger, reproduced the diuretic and in vivo protective effects of mannitol. Purified myoglobin infusion (35 mg) largely negated the beneficial action of mannitol. It was concluded that mannitol confers functional but not cytoprotection against the glycerol acute renal failure model, it acutely worsens renal bioenergetics, and its protective influence is probably due to a diuretic, not an antioxidant, effect.

Adaptive regulation of Na+‐dependent phosphate transport in the bovine renal epithelial cell line NBL‐1

1. Na(+)-dependent phosphate transport activity from bovine brush border membrane vesicles (BBMV) was reconstituted into artificial phospholipid membranes. It was shown, using lectin affinity chromatography, that transport activity was mediated by a glycoprotein. 2. The bovine kidney epithelial cell line NBL-1 expresses a Na(+)-dependent phosphate transporter which shows adaptive regulation in response to various media phosphate concentrations. Cells incubated in low-phosphate medium and medium containing 10 mM phosphate have Vmax values of 6.34 +/- 0.18 and 2.95 +/- 0.1 nmol.mg-1.3 min-1 and Km values of 17.1 +/- 3.0 and 36.3 +/- 7.14 microM for phosphate transport, respectively. Thus there is a significant (P less than 0.001) decrease in the Km and increase in the Vmax of transport when cells are transferred from 10 mM phosphate medium to low-phosphate medium. 3. Adaptation of phosphate transport in NBL-1 cells to low-phosphate medium was abolished by tunicamycin, an inhibitor of protein glycosylation, indicating that the transporter in these cells, like that in bovine BBMV, is a glycoprotein. 4. Cells were adapted to low-phosphate medium in the presence of [3H]mannose. Under these conditions a single membrane protein of molecular mass 55 kDa was labelled in the absence but not the presence of tunicamycin. Little labelling occurred if the cells were maintained in high-phosphate medium. 5. On the basis of these observations, it is proposed that the phosphate transporter of NBL-1 cells is a 55-kDa glycoprotein. Phosphate is accumulated across the brush border membrane of renal proximal tubule cells via a Na+/phosphate cotransport system. Na(+)-dependent phosphate transport has been extensively studied in isolated brush border membrane vesicles from rabbit and rat kidney [1-3]. The symport of phosphate and Na+ is electroneutral with a stoichiometry of 2Na+/phosphate at physiological pH values.

Hymenolepis diminuta: Further characterization of the membrane-bound acid phosphatase activity associated with the brush border membrane of the tapeworm's tegument

The acid phosphate activity (APA) associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzed p-nitrophenyl phosphate (PNPP), pyrophosphate ( PP i), and β-glycerophosphate (βGP). Inhibition of PNPP hydrolysis at pH 4.0 was inhibited in a competitive manner by the following compounds (listed in order of decreasing affinity with their apparent inhibitor constants ( K′ i )): molybdate (0.031 m M); PP i (0.147 m M); NaF (0.150 m M); o-carboxyphenyl phosphate (0.261 m M); inorganic phosphate (0.770); arsenate (3.45 m M); tartrate (22.1 m M); and βGP (29.8 m M). Cu 2+, formaldehyde, and arsenite at 10:1, 80:1, and 200:1 inhibitor to substrate ratios did not inhibit APA. The maximal rate of hydrolysis ( V max) of each substrate was greater at pH 4.0 than 5.0. The apparent Michaelis constant ( K′ m ) for PNPP increased from 0.233 to 0.351 m M when the pH was raised from 4.0 to 5.0. The K′ m for PP i decreased from 0.101 to 0.046 m M, while the K′ m for βGP changed from 2.04 to 2.22 m M under similar circumstances. APA and alkaline phosphatase activity increased as a function of temperature up to 45 °C.

Lactose-enhanced Uptake of Calcium by Isolated Brush Border Membrane Vesicles from the Rat Small Intestine.

Lactose-enhanced Uptake of Calcium by Isolated Brush Border Membrane Vesicles from the Rat Small Intestine.

Induction of a phosphomannosyl binding lectin activity inGiardia

Giardia lamblia, a protozoan parasite that causes widespread diarrheal disease, expresses a surface membrane associated lectin, taglin, which is specifically activated by limited proteolysis with trypsin, a protease that is present in abundance at the site of infection. When activated, taglin agglutinates enterocytes which are the cells to which the parasite adheres in vivo, and in addition, binds to isolated brush border membranes of these cells. These findings suggest that this lectin may be involved in the host-parasite interaction. Taglin is most specific for terminal phosphomannosyl residues and its binding to red cells is mediated by cell surface phosphate residues. Hemagglutinating activity induced by taglin is most active at pH 6.5 and is dependent on divalent cations. A monoclonal antibody to taglin reacts with the surface membrane of live trophozoites and recognizes a protein of 28/30 kDa in lysates of Giardia trophozoites, by immunoblotting. This finding is confirmed by direct demonstration of lectin activity by erythrocyte binding to proteins electroblotted to nitrocellulose, which revealed specific red cell binding to giardial protein bands in the same molecular weight range as those recognized by the monoclonal antibody.

Acute and chronic exposure to ethanol and the electrophysiology of the brush border membrane of rat small intestine.

In this study we have investigated the effects of (a) chronic ethanol intake on glucose and galactose absorption across the rat jejunum in vivo and on the potential difference across the isolated brush border membrane (Vm) and (b) acute exposure to ethanol (4% or 8%) and acetaldehyde (0.25%) on changes in Vm associated with Na(+)-dependent galactose absorption across the jejunum and ileum. Chronic ethanol intake was associated with hyperpolarization of Vm and an enhanced galactose but not glucose transport. Acute ethanol and acetaldehyde were without effect on Vm whether or not galactose was present. We conclude that while a greater electrochemical gradient across the brush border membrane is a likely explanation for the stimulation of galactose absorption induced by ethanol feeding, factors other than changes in Vm are responsible for the inhibitory effects of acute ethanol.

Cadmium inhibition of l-alanine transport into renal brush border membrane vesicles isolated from the winter flounder ( Pseudopleuronectes americanus)

Using isolated brush border membrane vesicles from the kidney of the winter flounder ( Pseudopleuronectes americanus), we have studied the effect of cadmium on l-alanine transport. Pretreatment of vesicles with 0.1 m m Cd 2+ resulted in inhibition of l-alanine uptake in the presence of a NaCl (but not KCl) gradient. Inhibition was due to a specific interaction with the sodium-alanine cotransport system and not a change in the driving forces for alanine transport, since Cd 2+ did not affect sodium-dependent d-glucose uptake. The effect of Cd 2+ on Na +-alanine cotransport showed mixed-type inhibition which is only partially reversible by EDTA. Cd 2+ uptake itself was shown to be time and temperature dependent, resulting in binding to both sides of the membrane. No direct correlation was possible between inhibition of l-alanine transport and the amount of Cd 2+ taken up by the membranes. Nevertheless, the striking time dependence of the effect of Cd 2+ on sodium-dependent l-alanine uptake and the inability of EDTA to reverse the inhibitory action of Cd 2+ suggest that Cd 2+ inhibits Na +-alanine cotransport at the cytoplasmic side of the membrane.

Electroneutral, HCO3--independent, pH gradient-dependent uphill transport of Cl- by ileal brush-border membrane vesicles. Possible role in the pathogenesis of chloridorrhea

By applying a rapid filtration technique to isolated brush border membrane vesicles from guinea pig ileum, 36Cl uptake was quantified in the presence and absence of electrical, pH and alkali-metal ion gradients. A mixture of 20 mM-Hepes and 40 mM-citric acid, adjusted to the desired pH with Tris base, was found to be the most suitable buffer. Malate and Mes could be used to replace the citrate, but succinate, acetate and maleate proved to be unsuitable. In the absence of a pH gradient (pHout:pHin = 7.5:7.5), Cl- uptake increased slightly when an inside-positive membrane potential was applied, but uphill transport was never observed. A pH gradient (pHout:pHin = 5.0:7.5) induced both a 400% increase in the initial Cl- influx rate and a long-lasting (20 to 300 s) overshoot, indicating that a proton gradient can furnish the driving force for uphill Cl- transport. Under pH gradient conditions, initial Cl- entry rates had the following characteristics. (1) They were unaffected by cis-Na+ and/or -K+, indicating the absence of Cl-/K+, Cl-/Na+ or Cl-/K+/Na+ symport activity. (2) Inhibition by 20-100 mM-trans-Na+ and/or -K+ occurred, independent of the existence of an ion gradient. (3) Cl- entry was practically unaffected by short-circuiting the membrane potential with equilibrated potassium and valinomycin. (4) Carbonyl cyanide m-chlorophenylhydrazone was strongly inhibitory and so, to a lesser extent, was 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid [(SITS)], independent of the sign and size of the membrane potential. (5) Cl- entry was negligibly increased (less than 30%) by either trans-Cl- or -HCO3-, indicating the absence of an obligatory Cl-/anion antiport activity. In contrast, the height of the overshoot at 60 s was increased by trans-Cl-, indicating time-dependent inhibition of 36Cl efflux. That competitive inhibition of 36Cl fluxes by anions is involved here is supported by initial influx rate experiments demonstrating: (1) the saturability of Cl- influx, which was found to exhibit Michaelis-Menten kinetics; and (2) competitive inhibition of influx by cis-Cl- and -Br-. Quantitatively, the conclusion is warranted that over 85% of the total initial Cl- uptake energized by a pH gradient involves an electroneutral Cl-/H+ symporter or its physicochemical equivalent, a Cl-/OH- antiporter, exhibiting little Cl- uniport and either Cl-/Cl- or Cl-/HCO3- antiport activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular aspects of proximal tubular phosphate reabsorption

In vivo manipulations to alter renal Pi reabsorption and the subsequent isolation of proximal tubular brush border membrane vesicles have greatly increased our knowledge about the regulation of renal Pi reabsorption via the Na+/Pi cotransport system. Only recently, direct biochemical and cell-biological access has become possible by the use of established and primary cell cultures. Based on the results obtained with isolated brush border membranes and cultured cells, a model has been presented, which might serve as a basis for future research of the regulatory control mechanisms of the renal Na+/Pi cotransport. At present, a major drawback is the fact that the molecular identity of the Na+/Pi cotransport system is still unknown. The identification of this transport system would certainly be a great step and would allow to verify or falsify one or the other hypotheses postulated in the past few years for the regulatory control mechanism(s) of the renal Na+/Pi cotransport.

Preliminary Studies on Attachment of the ‘Bacteroides fragilis group' to Porcine Intestinal Brush Borders

Adhesion was studied by phase contrast microscopy, scanning (SEM), and transmission (TEM) electron microscopy. Twenty-three of 26 Bacteroides fragilis strains adhered to the isolated brush border membranes. The percentage of bacterial cells that adhered varied between strains from 10 to 38. Seven of 25 strains belonging to the other species within the ‘ Bacteroides fragilis group’ contained cells that adhered. The attached bacteria showed no outer appendages or outer ruthenium red positive material when examined by SEM and TEM. Keywords: Bacteroides fragilis ; Intestinal brush border membranes; Adhesion.

Influence of insulin on phosphate uptake by brush border membranes from human placenta

Regulation of phosphate transport by insulin was investigated in brush border membranes from human placenta at term. At 22°C, a 45 min incubation of the total tissue with 10 −6 M insulin significantly decreased both the initial rate and the peak of sodium-dependent phosphate uptake by the corresponding brush border membranes. In contrast, Na + transport was not influenced by the hormone. Increasing the insulin concentration from 0 to 10 −5 M resulted in a dose-dependent inhibition of phosphate uptake with half-maximal effect at 1.1 × 10 −9 M. The hormone decreased PO 4 transport by decreasing the affinity of the carrier for the substrate ( K m = 0.180 ± 0.010 mM and 0.215 ± 0.015 mM in absence and presence of 10 −6 M insulin respectively, P < 0.05). The inhibitory effect of insulin required the presence of Mn 2+ whereas neither Mn 2+ nor insulin alone had any influence on PO 4 uptake. It is therefore assumed that receptor phosphorylation, which needs the presence of Mn 2+, is an intermediate step of insulin action on PO 4 uptake by the subsequently isolated brush border membranes. In contrast, insulin had no effect on PO 4 uptake when the membranes were directly incubated with the hormone prior to the transport measurement, suggesting that an intracellular messenger is needed for the inhibitory effect. This messenger is not cAMP since insulin at 10 −6 M concentration has no effect on cAMP content of the total placental tissue. Since insulin binding occurs predominantly if not exclusively on the brush border membrane facing the maternal blood compartment, it is assumed that only the maternal insulin influences the phosphate transport through the placenta.

Identification of proline-specific carboxypeptidase localized to brush border membrane of rat small intestine and its possible role in protein digestion.

A proline-specific carboxypeptidase (carboxypeptidase P, EC 3.4.12.-) was identified and partially characterized in the brush border membrane fraction of rat intestinal enterocytes and shown to be distinct from pancreatic proteases. The carboxypeptidase activity of isolated brush border membranes, with Z-Gly-Pro-Leu as substrate, was 43 nmol/min/mg protein representing a 16-fold purification when compared with mucosal cell homogenates. Activity was maximal in the middle region of the small intestine, and villus cells had twice the activity of crypt cells. Carboxypeptidase activity was maximal at pH 7.0, was stimulated by divalent cations, and was inhibited by metal chelating agents, suggesting that it is a metalloenzyme. The enzyme had the highest activity with synthetic peptides containing proline penultimate to the carboxy terminus. In vivo patterns of hydrolysis and absorption of amino acids from Z-Pro-Trp were examined using an intestinal perfusion technique. These studies indicate that brush border membrane carboxypeptidase may play an important role in the digestion of proline-containing peptides and proteins.

Inhibition of cholesterol absorption in rats by plant sterols.

The extent and site(s) of inhibition of cholesterol absorption by plant sterols, sitosterol and fucosterol, were studied in rats. The intragastric administration of a single emulsified lipid meal containing 25 mg [3H]cholesterol and 25 mg of either sitosterol or fucosterol inhibited the lymphatic absorption of cholesterol by 57% and 41%, respectively, in 24 hr. Less than 2% of each plant sterol was absorbed in the 24-hr period. In contrast, neither plant sterol (50 microM) inhibited cholesterol absorption when co-administered with equimolar amounts of cholesterol in phospholipid-bile salt micelles nor was either absorbed from the micellar solution. A series of in vitro studies was conducted to identify the site(s) of plant sterol inhibition of cholesterol absorption and to account for the difference in inhibitory effectiveness of sitosterol and fucosterol. A comparison of the micellar solubility of each sterol alone and in equimolar binary mixtures (to 2.0 mM) revealed that the solubility of individual sterols decreased in the following order: cholesterol, fucosterol, sitosterol, and that in binary mixtures cholesterol solubility was decreased by sitosterol and, to a lesser extent, by fucosterol relative to its solubility alone. A comparison between micellar-solubilized cholesterol and either sitosterol or fucosterol for binding to isolated brush border membranes, intestinal mucin, or for esterification by either cholesterol esterase or acyl coenzyme A:cholesterol acyltransferase revealed moderate to no competition. The data suggest that plant sterols displace cholesterol from bile salt (taurocholate) micelles and that sitosterol is more effective than fucosterol in this capacity.

Sodium-Dependent L-Lactate Uptake by Bovine Intestinal Brush Border Membrane Vesicles

In ruminants, intestinal digesta can contain considerable amounts of lactic acid derived from ingestion of lactic acid-containing feed and from production of lactic acid during ruminal digestion of readily fermentable carbohydrates. The aim of the present study was to investigate L-lactate transport across the bovine intestinal brush border membrane. The experiments were performed using isolated brush border membrane vesicles from the midjejunum of heifers. The results demonstrate the existence of Na+-stimulated L-lactate uptake by the brush border membrane vesicles. Acetate, propionate, and butyrate strongly inhibited Na+-dependent L-lactate transport. Acetate caused a 58% inhibition, whereas propionate and butyrate completely inhibited Na+-dependent uptake. Kinetic evaluation of L-lactate uptake in the presence or absence of extravesicular butyrate suggests a competitive inhibition by butyrate. Among the phenolic acids tested in this study only trans-cinnamic acid caused a significant reduction of L-lactate uptake, whereas cumaric acid, ferulic acid, and gallic acid only slightly reduced L-lactate transport. Thus, the L-lactate transporter appears to have some affinity for transcinnamic acid.

Mechanisms of absorption of inorganic mercury from rat small intestine. II. Composite effects of pH and halide ions on transport of mercuric chloride into isolated brush border membrane vesicles in rats.

Composite effects of pH and halide ions on the transport of HgCl2 into brush border membrane vesicles (BBMV) were investigated in rats. BBMV were incubated for 10 min. in buffer solution at different pH containing 10(-4) M HgCl2. The increase in pH increased the uptake of Hg by BBMV as a result of the increase in transport of Hg into intravesicular space and decrease in binding of Hg to BBMV. The isotonic displacement of NaCl in the buffer solution by LiCl or KCl did not change the uptake of Hg at each pH. The displacement of NaCl by mannitol increased the uptake of Hg at each pH, while the displacement by NaBr or NaI decreased the uptake and diminished the increasing effect of pH on the uptake of Hg. These changes in uptake of Hg due to the displacement were mainly ascribed to the changes in transport of Hg. These results suggest that the increase in pH mainly increases the transport of HgCl2 as a result of the conversion to hydroxide forms of Hg such as Hg(OH)Cl and Hg(OH)2, and Cl- Br- and I- act as the competing ions with OH- and decrease the transport of Hg.

Acid phosphatase activity in the isolated brush border membrane of the tapeworm, Hymenolepis diminuta: partial characterization and differentiation from the alkaline phosphatase activity.

The isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzes p-nitrophenyl phosphate over a broad pH range. Acid phosphatase activity (pH optimum at 4.0) is inhibited specifically by sodium dodecyl sulfate (SDS) and NaF, while the alkaline phosphatase activity (pH optimum at 8.8) is inhibited specifically by levamisole, 2-mercaptoethanol, and ethylenediaminetetra-acetate (EDTA). These two phosphatase activities are further differentiated in that (1) there is a rapid decrease in alkaline phosphatase activity when the membrane preparation is incubated at pH 4.0, while there is little loss of acid phosphatase activity, and (2) the alkaline phosphatase activity is solubilized with no loss of activity when the membrane is treated with Triton X-100, while such treatment causes a significant loss of acid phosphatase activity. Both activities are nonspecific and hydrolyze a variety of phosphorylated compounds, but the relative activities of the two phosphatases against these substrates vary significantly.

Evaluation of a Possible Functional Relationship between Chemical Structure of Intestinal Brush Border and Immunity to Trichinella spiralis in the Rat

Primary exposure to Trichinella spiralis in the rat, while immunizing against reinfection, induces changes in the carbohydrate structure of intestinal brush border membranes. Immunity is expressed in heightened resistance to mucosal invasion by L1 larvae, and the change in structure is evident in reduced membrane binding of the lectin, wheat germ agglutinin. The possibility that altered membrane composition is a requisite for expression of immunity was hypothesized and this was evaluated by correlating the maximum, specific binding of wheat germ agglutinin by isolated brush border membranes with (1) the expression of immunity acquired passively through serum transfer, and (2) the loss of immunity acquired from serial infections terminated in the intestinal phase. The hypothesis was further evaluated by determining whether the change in membrane structure represents a stimulus-specific response. We observed that (1) passively acquired immunity was not associated with a reduction in lectin binding and (2) short-term exposure to the intestinal stages of T. spiralis led to a reduction in lectin binding that was detectable at a time when rats were incapable of resisting reinfection. The change in lectin binding associated with trichinosis also accompanied infection with Nippostrongylus brasiliensis. Results uniformly support the conclusion that immunity to T. spiralis is independent of brush border membrane changes reflected in reduced binding of wheat germ agglutinin.

Transport of sulfate and phosphate in small intestine and renal proximal tubule: Methods and basic properties

1. 1. Isolated brush border membrane vesicles, basolateral membrane vesicles, and cultured renal epithelial cells provide good material for studying transport systems. 2. 2. The vesicle systems have been used to study the transport of labeled phosphate, sodium/phosphate cotransport, sodium/sulfate cotransport, basolateral transport of sulfate and basolateral transport of phosphate via anion exchange. 3. 3. Cultured renal cells show sodium/phosphate cotransport and parathyroid dependent inhibition of phosphate transport.