ATP-driven copper transport across the intestinal brush border membrane

The divalent metal ion transporter DMT1 is localized in the brush border membrane (BBM) of the upper small intestine and has been shown to be able to transport Mn2+, Fe2+, Co2+, Ni2+, and Cu2+. Belgrade rats have a glycine-to-arginine (G185R) mutation in DMT1, which affects its function. We investigated copper transport with BBM vesicles of Belgrade rats loaded with calcein, which exhibits fluorescence quenching by various metal ions. Transport of copper was disrupted in unenergized BBM vesicle of b/b Belgrade rats, as had been described for iron transport, while +/b vesicles exhibited normal transport by DMT1. When either b/b or +/b vesicles were loaded with ATP and magnesium, similar high-affinity accumulation of copper was observed in both types of vesicles. Thus, brush border membranes possess an ATP-driven, high-affinity copper transport system which could serve as the primary route for copper uptake by the intestine.     

Glycine uptake in brush border vesicles isolated from the rat intestinal cells

The uptake of glycine in osmotically active brush border membrane vesicles (obtained by the Mg++ precipitation method) has been studied and a partial characterization of its transport system has been established. The glycine uptake in these vesicles was stimulated by the presence of sodium and in the presence of an inwardly directed Na+ -gradient glycine was accumulated inside the vesicles. The effect of Na+ was specific; other monovalent cation as Li+, K+, Rb+ and choline were uneffective in the stimulation of glycine uptake, under the same experimental conditions. Preliminary experiments show an important role of some anions on the glycine uptake. A strong inhibition in the uptake rate was found when the measurements were carried out in the presence of sodium cyclamate, while in the presence of NaSCN the measured uptake values were similar to those observed in the presence of NaCl.     

The uptake of phosphatidylcholine by small intestinal brush border membrane is protein-mediated

Brush border membrane vesicles prepared from rabbit small intestine are essentially free of basolateral membranes and nuclear, mitochondrial, microsomal and cytosolic contaminants. The resulting brush border membrane is unstable due to intrinsic lipases and proteinases. The PC transfer between small unilamellar lipid vesicles or mixed lipid micelles as the donor and the brush border membrane vesicles as the acceptor is protein-mediated. After proteolytic treatment of brush border membrane with papain or proteinase K the PC transfer activity is lost and the kinetics of PC uptake are similar to those measured with erythrocytes under comparable conditions. Evidence is presented to show that the PC transfer activity resides in the apical membrane of the enterocyte and not in the basolateral part of the plasma membrane. Furthermore, the activity is localized on the external surface of the brush border membrane exposed to the aqueous medium with its active centre probably not in direct contact with the lipid bilayer of the membrane. Proteins released from brush border membrane by proteolytic treatment catalyze PC exchange between different populations of small unilamellar vesicles. Furthermore, these protein(s) bind(s) PC forming a PC-protein complex.     

Molecular organization of the intestinal brush border

The brush border of enterocytes represents one of the more specialized apical poles of epithelial cells. It is formed by particularly well-developed apical plasma membrane microvilli, whose shape is ensured by a highly organized cytoskeleton. The molecular organization of the cytoskeleton is described. Whereas several cytoskeleton proteins are ubiquitous, villin is highly specific for intestinal cells and can be used as a differentiation marker of these cells. The major glycoproteins, in particular hydrolases, of the brush border membrane have been characterized. They have many common structural features, in particular their mode of integration into the membrane by their N-terminal hydrophobic sequences that also plays the role of the 'signal peptide' responsible for their co-translational insertions into the endoplasmic reticulum. Studies on the biosynthesis and intracellular pathway of aminopeptidase N strongly suggest that sorting of apical and basolateral glycoproteins could occur after their integration into the basolateral domain.     

Hibernation enhancesd-glucose uptake by intestinal brush border membrane vesicles in ground squirrels

The ability to actively transport nutrients is maintained in intestinal tissues of hibernating ground squirrels compared with their active counterparts, and shows apparent upregulation in hibernators when transport rates are normalized to tissue mass. To identify the mechanisms responsible for the preservation of transport function during the extended fast of hibernation, we studiedd-glucose uptake into jejunal brush border membrane vesicles prepared from active and hibernating 13-lined ground squirrels. Hibernators were without food and showing regular bouts of torpor for at least 6 weeks before sacrifice. Electron micrographs indicated similar microvillus heights of jejunal enterocytes in the two activity states, whereas microvillus density was slightly greater in the hibernators. Glucose uptake into brush border membrane vesicles was inversely related to medium osmolarity, indicating negligible binding of substrate to brush border membrane vesicles surfaces, and intravesicular spaces were similar in hibernating and active squirrels. Glucose uptake showed strong Na⁺ dependency in both groups, with equivalent overshoot values in the presence of Na⁺. Kinetic analysis revealed a significant increase in the maximal velocity of transport (J _max) in hibernators (55.9±5.6 nmol·min^-1·mg^-1) compared with active squirrels (36.7±5.1 nmol·min^-1·mg^-1,P<0.05), with no change inK _m. Thus, the structure and absorptive capacity of the intestinal brush border persists in fasted hibernators, and the increase inJ _max for glucose uptake during hibernation likely contributes to the enhanced Na⁺-dependent glucose absorption previously observed at the tissue level.Abbreviations BBM brush border membrane(s) - BBMV brush border membranes vesicles - SGLT1 Na⁺-glucose transporter - 3-OMG 3-orthomethylglucose - J _max maximal velocity of transport - K _m transporter affinity for substrate - T _b body temperature     

Mechanism of neomycin stimulation of D-glucose uptake in rabbit intestinal brush border membrane

In order to study the effect of the antibiotic neomycin on the intestinal epithelium, D-glucose was used as a probe molecule and its transport into rabbit brush border membrane vesicles was measured by a rapid filtration method. Treatment of the epithelium with neomycin sulfate prior to the preparation of the brush border membrane enhanced the D-glucose uptake, whereas neutral N-acetylated neomycin did not. This action of neomycin was related to its polycationic character and not to its bactericidal action. No significant difference could be demonstrated between the protein content or disaccharidase-specific activities of the brush border fractions from treated or non-treated intestines. Electrophoretic protein patterns of SDS-solubilized membrane were not significantly different after neomycin treatment. To gain more information on the mechanism involved in the stimulation of D-glucose transport, experiments were conducted on phosphatidyl glycerol artificial membranes and the results compared with those obtained with brush border membrane. At a concentration of 10(-7) M, neomycin decreased the nonactin-induced K+ conductance by a factor of approx. 100. The membrane conductance was linearly dependent on the neomycin concentration and the conductance in 10(-2) M KCl was 10 times that in 10(-3) M KCl. The valence of neomycin was estimated, from the slope of these curves, to be between 6 and 4. In contrast, acetylated neomycin had no effect on the nonactin-induced K+ membrane conductance. Therefore, the effect of neomycin on artificial membrane is related to its 4 to 6 positive charges. It is proposed that the stimulation of sugar transport in brush border membrane is related to screening of the membrane negative charges by the positively-charged neomycin. Accumulation of anions at the membrane surface then occurs and their diffusion into the intravesicular space would increase the transmembrane potential which, in turn, stimulates the entry of D-glucose.     

Ca+2 transport across intestinal brush border membranes of the cichlid teleost Oreochromis mossambicus

Summary Brush border membranes were isolated from tilapia (Oreochromis mossambicus) intestine by the use of magnesium precipitation and differential centrifugation. The membrane preparation was enriched 17-fold in alkaline phosphatase. The membranes were 99% right-side-out oriented as indicated by the unmasking of latent glyceraldehyde-3-phosphate dehydrogenase and acetylcholine esterase activity by detergent treatment. The transport of Ca⁺² in brush border membrane vesicles was analyzed. A saturable and a nonsaturable component in the uptake of Ca⁺² was resolved. The saturable component is characterized by a K _m much lower than the Ca⁺² concentrations predicted to occur in the intestinal lumen. The nonsaturable component displays a Ca⁺² permeability too high to be explained by simple diffusion. We discuss the role of the saturable component as the rate-limiting step in transmembrane Ca⁺² movement, and suggest that the nonsaturable component reflects a transport mechanism operating well below its level of saturation.The authors wish to thank Tom Spanings for his superb organization of fish husbandry, and Maarten de Jong (Dept. of Physiology, Faculty of Medicine, University of Nijmegen) for making the automated stopped-flow apparatus available to us.     

Electrodiffusive magnesium transport across the intestinal brush border membrane of tilapia (Oreochromis mossambicus).

Mg2+ transport across the brush border of proximal intestinal epithelium of the teleost fish Oreochromis mossambicus was investigated, using 27Mg2+ to trace movement of Mg2+. Mg2+ uptake in brush border membrane vesicles was stimulated by a K+ diffusion potential (inside negative). Electrodiffusive Mg2+ transport obeyed simple Michaelis-Menten kinetics and was strongly temperature dependent, indicative of a carrier mechanism. The metal ion specificity of this electrodiffusive pathway (inhibition potency order: Co > Mn = Ni > La > Ca > Gd > Ba), predicts a specific role in Mg2+ transport. Competitive inhibition by Co(III) hexammine [Co(NH3)(6)(3+)] suggests that this transport system interacts with the solvated Mg ion. We propose that this novel transport system allows the uptake of Mg2+ across the apical brush border membrane, and is involved in transcellular Mg2+ transport. Consequently, the prevailing potential difference across the apical membrane represents a major driving force for intestinal Mg2+ absorption.     

Temperature-induced transitions of porcine intestinal brush border membranes

Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33°C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35°C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35°C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35°C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33°C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.     

Detergent-induced proteolysis of rabbit intestinal brush border vesicles

Proteolysis of brush border vesicle proteins is induced by detergent solubilisation. This proteolysis is selective in that some of the proteins are more susceptible than others. The rate of induced proteolysis is decreased by decreasing the temperature, has a minimum of about pH 6 and is effectively prevented by a combination of the protease inhibitors, EDTA, diisopropylfluorophosphate and iodoacetamide.     

Choline influx across the brush border of guinea pig jejunum

Choline uptake across the mucosal border of guinea pig jejunum was measured to determine the characteristics of this step in intestinal absorption. Unidirectional influx of [¹⁴C]choline appears to proceed primarily by a saturable, carrier-mediated process at low mucosal choline concentrations; at high concentrations (>4 mM) the influx rate is approximately linearly related to the mucosal choline concentration, suggesting that absorption by passive diffusion predominates. Influx was only minimally reduced by elimination of Na⁺ from the mucosal test solution or by reduction of the intracellular Na⁺ concentration. Preincubation of tissue samples with metabolic inhibitors or with ouabain did not markedly reduce influx. These results are consistent with a model of choline transport across the brush border membrane by a carrier-mediated mechanism which is similar to that involved in fructose absorption but different from the Na⁺-dependent mechanism which participates in active transport of sugar and amino acids. At low lumenal choline concentrations, influx into colonic mucosa is slower than in jejunum and appears to be attributed solely to simple diffusion.     

Coupled sodium-chloride influx across brush border of flounder intestine

Summary Measurements of the unidirectional influxes of Na and Cl from the mucosal solution into the epithelium (J _me ) of flounder intestine under short-circuit conditions reveal the presence of a coupled NaCl influx process at the brush border membrane which appears to be essential for the absorption of these ions.J _me ^Cl andJ _me ^Na were inhibited by replacing Na or Cl, respectively, in the bathing media with nontransported ions which also reduced the short-circuit current (I _sc) to near-zero values. Addition of furosemide to the mucosal solution alone inhibited theI _sc and reducedJ _me ^Cl andJ _me ^Na under control conditions, but not in the absence of Na or Cl, respectively. The reductions inJ _me ^Cl andJ _me ^Na elicited by ion replacement or furosemide were approximately equal, suggesting that the coupled influx mechanism mediates a one-for-one entry of these ions into the cell from the mucosal solution. Furosemide inhibited Cl absorption by reducing the unidirectional Cl flux from mucosa to serosa, consistent with its inhibition of the influx process. As in other epithelia, coupled NaCl influx is inhibited by cyclic AMP, which accounts for the decrease in Cl absorption elicited by cyclic nucleotides. These results support the notion thattranscellular NaCl transport is a neutral process and that the serosa-negative transepithelial electrical potential difference and preponderance of Cl over Na absorption under short-circuit conditions result from dissimilar permeabilities of the paracellular pathway to Na and Cl.     

Amphibian intestinal brush border enzymes during thyroxine-induced metamorphosis

Summary The development of intestinal brush border hydrolytic activities has been studied during thyroxine-induced metamorphosis of Rana catesbeiana. Alkaline phosphatase activity peaks at 3 and 10 days after the beginning of the thyroxine treatment. The cytochemical observations concerning alkaline phosphatase activity are in agreement with the biochemical data. At the ultrastructural level, alkaline phosphatase activity is particularly evident on the microvilli membranes of the enterocytes in the primary epithelium after 3 days and in the secondary epithelium after 10 days. -glutamyltranspeptidase exhibits an increase of activity between 7 and 10 days. On the other hand, glucoamylase, maltase, trehalase and leucylnapthylamidase activities decrease during thyroxine treatment, these enzymatic activities being lower than that normally observed after natural metamorphosis. The present study indicates that even though thyroxine is able to induce the morphological differentiation of the intestinal epithelium this hormone is unable to complete the enzymatic load of the new mucosa.This work has been supported by grants from France-Québec (M.D., J.H.) and from the Medical Research Council of Canada (D.M., J.S.H.)     

Amphibian intestinal brush border enzymes during thyroxine-induced metamorphosis

The development of intestinal brush border hydrolytic activities has been studied during thyroxine-induced metamorphosis of Rana catesbeiana. Alkaline phosphatase activity peaks at 3 and 10 days after the beginning of the thyroxine treatment. The cytochemical observations concerning alkaline phosphatase activity are in agreement with the biochemical data. At the ultrastructural level, alkaline phosphatase activity is particularly evident on the microvilli membranes of the enterocytes in the primary epithelium after 3 days and in the secondary epithelium after 10 days. gamma-Glutamyltranspeptidase exhibits an increase of activity between 7 and 10 days. On the other hand, glucoamylase, maltase, trehalase and leucylnapthylamidase activities decrease during thyroxine treatment, these enzymatic activities being lower than that normally observed after natural metamorphosis. The present study indicates that even though thyroxine is able to induce the morphological differentiation of the intestinal epithelium this hormone is unable to complete the enzymatic load of the new mucosa.