Brush Border Fraction Research Articles

Transferrin receptor distribution and regulation in the rat small intestine

Abstract A combination of biochemical quantitation and immunohistochemistry has been used to examine in detail transferrin receptor distribution and expression in the rat small intestine and its relationship to iron absorption. Receptor numbers were quantitated by transferrin binding to preparations of basolateral or brush-border membranes. Receptors were demonstrated on the basolateral membranes of the gut cells, but not on the brush-border fraction. Apotransferrin demonstrated little binding to basolateral membranes at physiological pH. Dietary or parenteral iron loading of animals produced a significant decline in transferrin binding, whereas binding was increased in iron deficiency. These data were confirmed by immunohistochemical studies using a monoclonal antibody to the transferrin receptor. When iron absorption was increased threefold following acute hemolysis and without a decrease in body iron stores, there was no change in transferrin receptor number. These data indicate that intestinal transferrin receptors may be regulated by body iron stores but suggest that they are not directly involved in iron absorption.

Transferrin receptor distribution and regulation in the rat small intestine: Effect of iron stores and erythropoiesis

A combination of biochemical quantitation and immunohistochemistry has been used to examine in detail transferrin receptor distribution and expression in the rat small intestine and its relationship to iron absorption. Receptor numbers were quantitated by transferrin binding to preparations of basolateral or brush-border membranes. Receptors were demonstrated on the basolateral membranes of the gut cells, but not on the brush-border fraction. Apotransferrin demonstrated little binding to basolateral membranes at physiological pH. Dietary or parenteral iron loading of animals produced a significant decline in transferrin binding, whereas binding was increased in iron deficiency. These data were confirmed by immunohistochemical studies using a monoclonal antibody to the transferrin receptor. When iron absorption was increased threefold following acute hemolysis and without a decrease in body iron stores, there was no change in transferrin receptor number. These data indicate that intestinal transferrin receptors may be regulated by body iron stores but suggest that they are not directly involved in iron absorption.

Simultaneous isolation and characterization of brush border and basolateral membrane vesicles from bovine small intestine.

Purified brush border and basolateral membranes were isolated from homogenized intestinal enterocytes of Holstein steers by divalent cation precipitation followed by differential and sucrose density gradient centrifugation. Alkaline phosphatase and Na/K adenosine triphosphatase served as marker enzymes for the brush border and basolateral membranes, respectively. The brush border and basolateral membrane fractions were enriched 5.1- and 10.1-fold, respectively, over the cellular homogenate. Electron micrographs, obtained with transmission electron microscopy, confirmed the vesicular nature of the membranes and revealed that basolateral membrane vesicles generally were smaller and more irregular in shape than brush border membrane vesicles. The vesicular nature of isolated membrane preparations was confirmed with osmotic activity experiments. Enrichment of brush border and basolateral membrane fractions compared to the initial homogenate and the vesicular configuration of both preparations indicate that the isolated brush border and basolateral membrane preparations were suitable models for evaluating nutrient transport properties of bovine small intestine. The number of transport experiments possible per animal using the membrane vesicle technique is many times more efficient than some other in vitro techniques (i.e., intestinal rings or everted sacs).

Thymocytes Reacting with Heterologous Antibodies against GP 330 in Autologous Immune Complex Glomerulopathy (AICG) in the Rat.The Relation between Susceptibility for AICG and Anti-GP 330-Binding Thymocytes

Autologous immune complex glomerulopathy (AICG) is induced by immunizing rats with a crude brushborder fraction of rat kidney tubules (Fx1A) or with the purified GP 330 antigen.In these animals, anti -brushborder antibodies develop, leading to subepithelial immune complexes along the glomerular capillary wall.In rats with AICG, thymocytes sensitized against Fx1A as well as thymocytes recognizing anti-Fx1A are present.These latter cells might play a role in the specific tolerance against the pathogenetic antigen GP 330.To substantiate this notion, immunofluorescence studies were performed in which the number of anti-GP 330 binding cells was quantified in thymus cell suspensions of rats with AICG, in control rats and in naive rats with different genetic background.It is shown that increased numbers of antiGP 330-binding thymocytes in rats with AICG are associated with a decline in the serum antibrushborder titer.Furthermore, it appears that the number of anti-GP 330-binding thymocytes in naive rats of the non-responder Brown Norway strain is significantly higher as compared to the PVG/c and Lewis strains, which are susceptible for AICG.The correlation between the numbers of anti-GP 330-binding thymocytes and the susceptibility for AICG suggests a role for these cells in maintaining the tolerance against the Fx1A (GP 330) antigen.

Further studies on the effect of aldosterone on Mg 2+-HCO 3−-ATPase and carbonic anhydrase from rat intestinal mucosa

The main purpose of this study is to elucidate the effect of adrenocorticoids on Mg 2+-HCO 3 −-ATPase and carbonic anhydrase which are thought to be related to anion transport in mammalian intestinal mucosa and renal tubulus. Rat duodenal mucosa, large intestinal mucosa and kidney cortex were excised and homogenized with mannitol-Tris buffer (pH 7.1) and brush border fraction and cytosol were obtained by a differential fractionation procedure. Brush border Mg 2+-HCO 3 −-ATPase and cytosol carbonic anhydrase activities in the duodenal mucosa decreased to 70% and 37% of normal values, respectively 5–11 days after adrenalectomy. Adrenalectomy also decreased significantly both enzyme activities in large intestinal mucosa; on the other hand, renal enzyme activities did not change. Four hours after a single injection of 20–80 μg/kg of aldosterone, ip, to adrenalectomized rats, Mg 2+-HCO 3 −-ATPase and carbonic anhydrase activities in duodenal mucosa increased gradually to normal or near normal in dose-dependent fashion. Both enzyme activities in large intestinal mucosa were also increased by a larger dose of aldosterone. Again, renal enzyme activities were not affected by any dose of aldosterone. In contrast, corticosterone (1 mg and 4 mg/kg) and dexamethasone (50 μg and 200 μg/kg) had no replacement effect on enzyme activities in all organs. These results showed that the mineralocorticoid, but not glucocorticoids, is a regulator of the enzyme activity of Mg 2+-HCO 3 −-ATPase and carbonic anhydrase from intestinal mucosa. The true mechanisms by which both enzymes are activated by aldosterone are not clear at present.

Conductive sodium pathway with low affinity to amiloride in LLC-PK1 cells and other epithelia.

Electrical potential driven 22Na+ fluxes were measured in membrane vesicles prepared from a number of cultured and naturally occurring epithelia. In all preparations a rheogenic pathway blocked by 200 microM (but not by 1.5 microM) amiloride was noted. This transporter was characterized in membranes prepared from cultured LLC-PK1 cells. In this preparation more than 50% of the rheogenic 22Na+ uptake was blocked by amiloride (IC50 approximately 30 microM), phenamil (IC50 approximately 66 microM), or ethylisopropylamiloride (IC50 approximately 5 microM). This amiloride-sensitive flux was not seen if the vesicles were partially depolarized by external Na+ or K+. It could not be driven by a pH gradient, did not require the presence of Ca2+, sugars, or amino acids, and showed little dependence on temperature (25 versus 0 degrees C). The data suggest the existence of an epithelial amiloride-blockable Na+ transporter different from the previously characterized Na+ channel, Na+/H+ and Na+/Ca2+ exchangers, and the Na+-hexose co-transporter. In rat kidney cortex membranes prepared by Mn2+ precipitation, this transporter is primarily located in the brush-border fraction.

Age-related alteration of renal brush border leucine aminopeptidase in rat kidney cortex

Renal brush border enzyme activities were significantly decreased with age. The decrease was observed in the homogenate and brush border fractions. Purified leucine aminopeptidase was significantly decreased in V max and K m value for leucyl-β-naphthylamide in the old rats. Heat stability of leucine aminopeptidase from both old and young rats showed biphasic, and the enzyme from old rats was more stable at 60°C than that of the young. However, other properties such as molecular weight, antigenicity, charge, and optimum pH were not significantly different between young and old rats. From these results, it is suggested that age-related alteration of leucine aminopeptidase is due to a conformational change of enzyme molecule; the conformational change might occur at the active sites of the enzyme.

Transglutaminase Activity along the Rat Small Bowel and Cellular Location

A recent report indicates a relationship between human transglutaminase (TG) jejunal mucosa activity and celiac disease. We investigated the enzyme distribution along six consecutive small bowel segments of mucosa and tested TG activity on brush border membranes obtained from whole mucosa homogenate in Wistar rats. TG activity was significantly present in jejunal mucosa even if mostly detected in the distal part of small intestine. Our study indicates highest enzymatic activity in the subcellular fraction containing organelles and cellular membranes (66.8%) while a 7% activity was associated with the brush border fraction.

Intestinal lactase and other disaccharidase activities of a suckling crabeater seal ( Lobodon carcinophagus)

1. 1. The intestinal disaccharidase activities of a suckling crabeater seal were investigated. 2. 2. Lactase, maltase, isomaltase and cellobiase activities were readily detected but trehalase and sucrase activities were absent. 3. 3. The intestinal homogenates were separated into a soluble (S2) fraction and a particulate brush border (P2) fraction. The lactase activities of the two fractions had different properties corresponding to those of an acid and a neutral β-galactosidase respectively. Approximately two-thirds of the total lactase activity measured at pH 6.0 was due to the acid β-galactosidase. 4. 4. The isomaltase and cellobiase activities were found almost exclusively in the particulate fractions but about one third of the maltase activity was in the S2 fraction. This soluble maltase activity appeared to be due to an acid maltase.

Effect of monensin on cell ultrastructure and glycoprotein migration in adult mouse jejunal epithelium in organ culture.

Explants from adult mouse jejunum were cultured for 3 h in a medium which contained both 3H-fucose (10 or 25 microCi/ml) and monensin (100 microM) or 3H-fucose only (control). Radiochemical analysis of cell fractions showed that 3H-fucose labelling of the brush border fraction decreased 42% in monensin-treated explants, suggesting that in absorptive cells the intracellular transport of newly synthesized glycoproteins to the apical plasma membrane had been inhibited. Electron-microscopic examination of treated explants revealed a variation in response to the drug from region to region. In some areas, both absorptive and goblet cells exhibited little alteration. In others, the Golgi cisternae of both absorptive and goblet cells were entirely replaced by large vacuoles, and in the latter cell type, the cisternae of the rough endoplasmic reticulum were greatly distended. Electron-microscopic radioautographic analysis showed that in absorptive and goblet cells exhibiting little morphological change, intracellular transport of newly synthesized glycoproteins was similar to that in controls. In regions where absorptive cells exhibited extensive Golgi modifications, intracellular transport remained normal in some cases; more often-however, there was a marked inhibition (over 70%) of transport of labelled glycoproteins to the apical surface. Transport to the basolateral membrane was never affected. In goblet cells exhibiting modifications of the Golgi apparatus and rough endoplasmic reticulum, no incorporation of 3H-fucose label in the Golgi apparatus occurred, suggesting a block of intracellular transport proximal to the site at which 3H-fucose is added. In absorptive cells, this does not appear to be the case, since the level of 3H-fucose incorporation in all treated cells remained similar to that in controls.

Localization of dehydropeptidase-I, an enzyme processing glutathione, in the rat kidney.

An antibody prepared against dehydropeptidase-I, one of the glutathione processing enzymes, from the renal membrane fraction of rats was employed to localize at the light microscopic level the enzyme in the kidney using the avidin/biotin-peroxidase complex method. Dehydropeptidase-I was found to be present on both the brush border and the basolateral membranes of proximal tubular cells. Furthermore, the enzyme activity in the isolated brush border and basolateral membrane fractions was also determined. Distribution profiles of the enzyme activity showed good agreement with the results of the immunohistochemical observations.

Age-related change in brush borders of rat kidney cortex

Age-related change of rat renal brush borders was examined with electron microscopy and biochemical procedures. Total activity of renal brush borders enzymes, such as alkaline phosphatase and leucine aminopeptidase in the homogenate, was significantly decreased wiht age. Acid phosphatase activity and protein content were not significantly changed with age. Specific activity of leucine aminopeptidase in brush border fraction was significantly decreased at a later stage of age. Protein content of brush border fraction was decreased significantly with age. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, some of the proteins disappeared during aging. Electron microscopic observations of kidney cortex showed that microvilli of renal brush borders from old rats were observed to be fewer than those from the young; epithelial cells in young rats have more densely packed brush border projections than those in old rats. From these results, it is suggested that during aging renal brush borders are degraded, and that protein components of the brush borders were different between old and young.

Human intestinal folate conjugase: adaptation after jejunoileal bypass

During intestinal absorption, dietary polyglutamyl folates are hydrolyzed to monoglutamyl folates by pteroylpolyglutamate hydrolase (folate conjugase). This enzyme is present in the brush border and intracellular fractions of human jejunal mucosa. We compared the activities of brush border and intracellular folate conjugase (BBFC and ICFC), and other mucosal enzymes in the jejunum in continuity and bypassed loop in nine patients who underwent intestinal reconstitution operations 3 to 9 yr after jejunoileal bypass. Control jejunum was obtained from seven obese patients undergoing gastric bypass surgery. Jejunal morphometry showed mucosal hyperplasia with taller villi, larger crypts, and no change in cell size in the jejunum in continuity as compared to control or bypass jejunum. In brush border fractions, specific activities of sucrase and BBFC were significantly greater in the jejunum in continuity than in the control or bypass jejunum. In contrast, the specific activities of the other brush border and intracellular enzymes, including ICFC, were similar in all three segments. This is the first evidence that BBFC, like sucrase, adapts to luminal nutrition. The difference in response of BBFC and ICFC to diet and/or pancreato-biliary secretions provides evidence that these are distinct enzymes which are regulated by different mechanisms.

Enterocytic differentiation and glucose utilization in the human colon tumor cell line Caco-2: modulation by forskolin.

The human colon cancer line Caco-2 exhibits after confluency a concomitant increase of glycogen accumulation and an enterocytic differentiation. The purpose of this work was to investigate whether forskolin (FK), an activator of adenylate cyclase, would induce a permanent glycogenolysis and, if so, whether it would result in modifications of the differentiation pattern of the cells. FK activates adenylate cyclase in Caco-2 cells with an ED50 of 7 X 10(-6)M. Three different treatment protocols with FK (10(-5)M) were applied: 1) the cells were treated during all the time in culture (20 days); 2) the treatment was started after confluency; 3) the treatment was interrupted after confluency. The presence of FK results in a permanent stimulation of cAMP accumulation (10 to 20 fold the basal values) and in a permanently reduced glycogen content (30 or 50% of the control values). The rates of glucose consumption are increased three and five fold in protocols 1 and 3 respectively. These metabolic changes are associated with morphological changes (tightening of the intercellular spaces and shortening of the brush border microvilli) and with a dual inhibition of the activities of brush border hydrolases: a) an inhibition of the post-confluent increase of activity of sucrase, aminopeptidase N and alkaline phosphatase in the brush border enriched fraction; b) an inhibition of the post-confluent increase of activity of sucrase in the cell homogenate. A comparison of the results obtained in each protocol shows that the morphological modifications and the decrease of the enzyme activities in the brush border fraction are regularly associated with an increased cAMP accumulation, whereas the inhibition of the differentiation of sucrase is a direct consequence of the increase in glucose consumption and decrease in glycogen stores.

Hormonal and drug effects on the degradation of human myelin basic protein peptide 43–88 by alkaline proteolytic activity in the rat kidney

The microsomal brush-border fraction of rat renal tissue contains enzymatic activity, optimally active at pH 9, that is capable of degrading human myelin basic protein (BP) peptide 43–88. In the present study, this degradation and the effect on it selected drugs and hormones were examined further. Of the substances tested, 10 −2M chloroquine and 10 −5M ACTH 1–24 were found to be the most effective inhibitors followed by 10 −5M ACTH 1–39; parathormone, glucagon and insulin were found to be inhibitors an order of magnitude weaker than ACTH 1–24. Hydrocortisone, dexamethasone, maleic acid and ACTH 4–10 were found to have minimal or no inhibitory effect on the peptide degrading activity. Gel filtration of the degradation products indicated that the rate of degradation of BP peptide 43–88 at pH 9 had been retarded by ACTH 1–24. These studies indicate that the clearance and catabolism of this peptide may be altered by available therapeutic agents.

Rat kidney brush border enzyme activity following subchronic oral lead exposure

Rats received 0.1% lead acetate in their drinking water for 3 weeks or for 6 weeks, at which time renal brush border fractions were obtained for measurement of enzyme activity. Renal brush border preparations from Pb 2+-exposed rats exhibited statistically significant decreases in the activity of γ-glutamyl transpeptidase and alanine aminopeptidase after 3 or 6 weeks of treatment. There was an increase in the activity of alkaline phosphatase which was statistically significant after 3 weeks of Pb 2+ exposure. The (Na +,K +) adenosine triphosphatase activity and urokinase activity, located in the basolateral membrane fractions, were unchanged by Pb 2+ exposure, as were the protein and phospholipid contents of the brush border fractions. The results are compared to those following acute exposure to Pb 2+ or Cd 2+.

Properties of an ecto-5'-nucleotidase of the renal brush border.

A decrease of glomerular filtration rate can be observed during accelerated catabolism of ATP in kidney. It has been proposed that this effect is due to the increase in the renal production of adenosine from ATP. The last reaction in the pathway concerned is the conversion of 5'-AMP to adenosine. We found that brush border membranes purified from homogenates of the rat renal cortex carry out this reaction. The enzyme involved in the hydrolysis has the characteristic properties of ecto-5'-nucleotidases: It is inhibited by ATP, ADP, and by alpha, beta-methyleneadenosine-5'-diphosphate, and it is not stimulated by magnesium. All catalytic sites are accessible from the outside of the vesicles. The Km of the enzyme for 5'-AMP is 5.77 microM. The enrichment of the 5'-AMP-hydrolyzing activity in the brush border fraction as compared to the homogenate is 9.2 +/- 1.5 times. Histochemical staining of kidney sections reveals hydrolysis of 5'-AMP only at the brush border of the proximal tubule. We conclude that the brush border of the proximal tubule of the rat kidney possesses an ecto-5'-nucleotidase which has the same properties as the ecto-5'-nucleotidases in other tissues.

Glycosaminoglycans of tegumental fractions of Hymenolepis diminuta

The teguments of 6 and 10 day-old Hymenolepis diminuta were removed with Triton X-100 and separated into brush border and vesicular fractions by differential centrifugation. Glycosaminoglycans (GAG) isolated from these tissues and from the denuded carcass were treated with specific GAG-degrading enzymes and other chemical agents and analyzed by sodium dodecyl sulfate-polyacrylamide, agarose gel and cellulose acetate electrophoresis. Both 6 and 10 day-old worm carcases contained chondroitin sulfate, heparin/heparan sulfate and hyaluronic acid. The 10 day-old worm brush border and vesicle fractions contained chondroitin sulfate but no heparin-like material. Colorimetric analysis showed that the carcasses of both 6 and 10 day-old worms contained uronic acid. About 98% of the detectable uronic acid of 10 day-old worms was found in the carcass, and only 2% in the brush border fraction. No uronic acid was detected in the other tegumental fractions.

Presence of an extensive clathrin coat on the apical plasmalemma of the rat kidney proximal tubule cell.

The nature of the cytoplasmic coat present on the apical invaginations of the kidney proximal tubule cell was investigated by immuneoverlay and immunocytochemistry of renal brush borders with anticlathrin antibodies. When kidney cortex was prepared for electron microscopy using methods that enhance visualization of clathrin coats, the apical invaginations at the base of the brush border microvilli were seen to be backed by a nearly continuous coating which resembles but is more extensive than the lattice-like clathrin coats found around brain coated vesicles. When isolated brush border fractions were prepared under conditions that preserve the coats, separated by SDS PAGE, and transferred to nitrocellulose, the presence of clathrin heavy and light chains was detected by immuneoverlay using two different affinity-purified anticlathrin IgGs--one that we prepared, which detects only the clathrin light chains, and the other, prepared by Louvard et al. ( Louvard , D., C. Morris, G. Warren, K. Stanley, F. Winkler , and H. Reggio , 1983, EMBO [Eur. Mol. Biol. Organ.] J., 2:1655-1664), which detects both the heavy and light chains. As viewed by light microscopy (immunofluorescence or immunoperoxidase), staining with both anticlathrins was concentrated at the base of the proximal tubule microvilli. Immunoelectron microscopic localizations carried out on brush border fractions (using peroxidase and gold conjugates) demonstrated specific binding of anticlathrin IgGs to the lattice-like cytoplasmic coat. When brush border fractions were reacted with monoclonal antibodies prepared against gp330 and maltase, proteins that serve as markers for the membrane of the apical invaginations and microvilli, respectively ( Kerjaschki , D., L. Noronha - Blob , B. Sacktor , and M. G. Farquhar , 1984, J. Cell Biol., 98:1505-1513), the two proteins retained their restrictive distribution in the brush border. The findings demonstrate (a) that the cytoplasmic coat of the proximal tubule intermicrovillar apical invaginations is composed of clathrin heavy and light chains, and (b) that the differential distribution of proteins in these two brush border microdomains is maintained in appropriately prepared brush border fractions.

Comparative study of some intestinal brush border membrane enzymes during perinatal development of the mouse

1. 1. Intestinal brush border membrane (bbm) fractions have been isolated from fetal and neonatal mice. 2. 2. The existence of discordant developmental patterns of intestinal enzymatic activity derived from total homogenate and bbm fraction was confirmed. 3. 3. It originates chiefly from two phenomena: (a) variations in the state of purity of brush border fractions, and (b) loss of brush border membrane enzyme activities in supernatant that increases with age. 4. 4. The phenomenon of solubility for glucoamylase and alkaline phosphatase is already present two days before birth.