Transport ofl-lysine by rat intestinal brush border membrane vesicles

Brush border membranes were isolated from rat jejunum by a divalent cation precipitation method.³H-l-Lysine uptake was measured by a rapid filtration technique. Uptake after prolongued incubation periods was osmotically insensitive and represented almost exclusively binding to the vesicles. Extrapolating initial linear uptake to a zero incubation time indicated no binding of the amino acid to the external membrane surface.Sodium did not significantly alter the initial uptake rate.l-Lysine transport respresents a carrier mediated uptake in the presence and absence of sodium as indicated by the transstimulation experiments. The transport mechanism operates stereospecifically and is inhibited by other basic amino acids andl-leucine andl-phenylalanine. Saturation experiments result in aK _m of 0.26 mmoles/l and aV _max of 272 pmoles/mg protein/10 s.Inside negative anion diffusion potentials and inside negative potassium diffusion potentials (valinomycin) were unable to increase the transport rate. Transmembrane pH-gradients were also unable to alter transport.Abbreviations HEPES N-2-hydroxethylpiperazine-N-ethane sulfonic acid - Tris tris(hydroxymethyl)aminomethane - EGTA ethyleneglycolbis-(-aminoethyl-ether)-N,N tetraacetic acid - MES 2-(N-morpholino)-ethanesulfonic acid     

Phosphate uptake by superficial and deep nephron brush border membranes

Dietary phosphate restriction and acute parathyroidectomy in rat are known to be associated with a selective increase in phosphate uptake by renal cortical brush border membranes (BBM). Conversely, phosphate loading and parathyroid hormone (PTH) administration result in a decrease of this uptake.In the present study, we investigated whether the response of the membrane to these various stimuli implies similar or different modifications of the kinetic properties of this membrane, whether these modifications affect one or both of the two systems of phosphate transport previously described, whether both superficial and deep nephron populations are involved, and whether the two stimuli: dietary phosphate, and parathyroid activity, are additive or not.Kinetic studies of phosphate (PO₄) uptake by BBM vesicles were performed in seven groups of rats: control (N), acutely thyroparathyroidectomized (TPTX), PTH loaded (PTH), phosphate loaded (P⁺), phosphate depleted (P^–) phosphate depleted with acute thyroparathyroidectomy (P^–TPTX), and phosphate depleted-PTH loaded (P^–PTH). In each of these experimental conditions, superficial and deep nephrons were investigated.Results indicate that 1. BBM from deep nephrons present a greater capacity for PO₄ transport than those from superficial nephrons; 2. Whereas a dual system of PO₄ uptake is observed in superficial BBM, deep BBM present only one single system; 3. Phosphate in the diet influences PO₄ uptake by BBM to a greater extent in the deep than in the superficial nephrons; 4. PTH status on the contrary, equally influences both populations; 5. TPTX does not significantly enhance PO₄ uptake in phosphate depleted rats; however, PTH loading curtails this uptake; 6. In the deep BBM neither the PTH status nor the phosphate content of the diet modify the apparentK _m. In the superficial BBM, the apparentK _m of the high affinity system (low substrate concentrations) varies with the PTH activity.     

Involvement of an enterocyte renin–angiotensin system in the local control of SGLT1-dependent glucose uptake across the rat small intestinal brush border membrane

There is increasing evidence that locally produced angiotensin AII (AII) regulates the function of many tissues, but the involvement of enterocyte-derived AII in the control of intestinal transport is unknown. This study examined whether there is a local renin–angiotensin system (RAS) in rat villus enterocytes and assessed the effects of AII on SGLT1-dependent glucose transport across the brush border membrane (BBM). Gene and protein expression of angiotensinogen, ACE, and AT₁ and AT₂ receptors were studied in jejunal and ileal enterocytes using immunocytochemistry, Western blotting and RT-PCR. Mucosal uptake of d -[¹⁴C]glucose by everted intestinal sleeves before and after addition of AII (0–100 n m ) to the mucosal buffer was measured in the presence or absence of the AT₁ receptor antagonist losartan (1 μ m ). Immunocytochemistry revealed the expression of angiotensinogen, ACE, and AT₁ and AT₂ receptors in enterocytes; immunoreactivity of AT₁ receptor and angiotensinogen proteins was especially pronounced at the BBM. Expression of angiotensinogen and AT₁ and AT₂ receptors, but not ACE, was greater in the ileum than the jejunum. Addition of AII to mucosal buffer inhibited phlorizin-sensitive (SGLT1-dependent) jejunal glucose uptake in a rapid and dose-dependent manner and reduced the expression of SGLT1 at the BBM. Losartan attenuated the inhibitory action of AII on glucose uptake. AII did not affect jejunal uptake of l -leucine. The detection of RAS components at the enterocyte BBM, and the rapid inhibition of SGLT1-dependent glucose uptake by luminal AII suggest that AII secretion exerts autocrine control of intestinal glucose transport.     

Effect of feeding a high protein diet on amino acid uptake into rat intestinal brush border membrane vesicles

Uptake of the neutral amino acidl-leucine into isolated rat intestinal brush border membrane (=BBM) vesicles and into a jejunal mucosa preparation as affected by the protein content of the diet was investigated. Adult rats fed either a high carbohydrate (HC) diet (11% protein) or a high protein (HP) diet (77% protein) for several weeks were used for the experiments.The time course ofl-leucine uptake into BBM vesicles prepared from the small intestine of HC-or HP-rats was studied under conditions of an inwardly directed Na⁺-gradient and under Na⁺-equilibrium conditions. Furthermore, in one series of experiments the Na⁺-equilibrium was replaced by a K⁺-equilibrium. l-leucine uptake under Na⁺-gradient conditions displayed the overshoot phenomenon typically associated with Na⁺-gradient-dependent active transport processes in BBM vesicles and the overshoot in group HP exceeded that in group HC significantly.Under both Na⁺-and K⁺-equilibrium conditionsl-leucine uptake into the BBM-vesicles also was faster in group HP.Finallyl-leucine uptake into jejunal mucosa in group HP exceeded that in group HC, too.The results therefore indicate that Na⁺-dependent and Na⁺-independent transport of neutral amino acids across the intestinal brush border membrane adapts to the dietary protein level.Some of the results were reported in a preliminary form [16]     

Rat intestinal brush border enzymes release by deoxycholate in vivo

The release of proteins, sucrase (SA), maltase (MA), leucine aminopeptidase (LA) and alkaline phosphatase (AP) activity from rat jejunum by sodium deoxycholate (DOC) was studied by an in vivo perfusion technique.In our experimental conditions, a 2 mmol/l DOC perfusion for 30 min induced a marked and reversible release of proteins and hydrolases. When specific activities were considered, each enzyme showed a distinct release pattern. Significantly, the SA release was largely increased, the AP release was decreased and there was no correlation between the releases of SA and AP. Furthermore, the various enzymes recovered into the lumen were solubilized at different extents. SA was chiefly present in a soluble and AP in a particular form.The microscopical appearances showed a slight exfoliation of the epithelial cells from the villous tips but no specific changes when compared to the control group.The results are discussed in terms of enzymic localization in the brush border membrane; SA would be located very superficially in the surface membrane and AP buried in the membrane and less accessible than the other enzymes.     

Oligopeptidases of brush border membranes of rat small intestinal mucosal cells

1. The properties of peptidases located in the brush borders of rat small intestinal mucosal cells have been investigated using a new method for the assay of peptidase activity. In this method amino acids produced by hydrolysis of peptides are oxidized by ophidian L-amino acid oxidase and the hydrogen peroxide produced during reoxidation of the reduced enzyme is estimated spectrophotometrically.2. 10-20% of the total cellular peptidase activity and 50% of the leucylnaphthylamidase (LNAase) activity were found to be tightly bound to the brush borders and to possess different substrate specificity from the supernatant peptidase activity.3. Evidence from kinetic and competition studies indicates the presence of more than one peptidase in the brush borders. The peptidases exhibit pH optima of 8.0-8.5, are inhibited by EDTA, but are not usually activated by divalent metal ions. The brush border peptidases hydrolyse di- and tripeptides, some oligopeptides, leucinamide and leucyl-beta-naphthylamine. On the basis of the Michaelis constants, these substrates differ in their affinities for the enzymes.4. It is proposed that the brush border peptidases serve an analogous function in the terminal stages of protein digestion to that of the disaccharidases in the case of carbohydrate digestion and absorption.     

Characterization of intestinal brush border cytoskeletal proteins of normal and neoplastic human epithelial cells. A comparison with the avian brush border.

The elaborate cytoskeletal matrix underlying the intestinal epithelial cell brush border (BB) is the hallmark of a mature enterocyte. As such, alterations in this structure are potentially useful as markers aiding in the recognition of subtle defects in cell maturation, such as those accompanying dysplasia and neoplasia. For exploration of this hypothesis, the BB components of human ileal and colonic enterocytes have been compared structurally and biochemically with the well-characterized avian BB, and alterations in the BB cytoskeleton in various states of dysplasia and neoplasia have been identified. Ultrastructural analysis of isolated human ileal BBs indicate that the human BB is structurally homologous to BBs isolated from chicken and other mammalian sources. Like other mammalian BBs (eg, from rat) the terminal web cytoskeleton of the human BB is less extensive than that in the avian BB. Immunochemical analysis of isolated human BBs indicates that the major proteins of the avian microvillar actin bundle, villin, fimbrin, and the 110-kd subunit of the 110K-calmodulin complex, are all present in the human BB. The terminal web protein myosin is also present. Unlike the terminal web of the avian BB, which contains a BB-specific isoform of spectrin, TW 260/240, the human BB contains the more widely distributed spectrin isoform, fodrin. In addition, the human BB contains multiple proteins immunoreactive with antibodies to protein 4.1, a spectrin/actin binding protein that is absent from the avian BB. Immunolocalization studies examining the distribution of the BB-specific microvillar protein, villin, in human colonic mucosa indicate that the localization of this protein is disrupted in certain dysplastic and neoplastic states. Thus, both the expression and/or distribution of BB-specific proteins such as villin may be useful markers for defects in the differentiation state of the enterocyte.     

Inorganic anion transport in kidney and intestinal brush border and basolateral membranes

The efflux of inorganic anions from purified brush border and basolateral membrane vesicles from dog kidney cortex was measured under equilibrium exchange conditions. Marked differences in temperature sensitivity and effects of inhibitors were found between the Cl and SO4 transport pathways and between the two types of membranes. SO4 transport in both brush border and basolateral membranes was markedly reduced by cooling, but significant inhibition by 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) was only observed in basolateral vesicles. In contrast, Cl efflux from both types of vesicles was neither substantially inhibited by DIDS nor by lowering the temperature to 0 degrees C. Phosphate efflux from basolateral membrane vesicles was found to be only partially sensitive to DIDS. Attempts to label the stilbene-sensitive SO4 pathway in basolateral vesicles using [3H2]DIDS as a marker were unsuccessful due to the nonspecific labeling of many membrane components. The asymmetry in inorganic anion transport behavior exhibited by brush border and basolateral membrane vesicles from dog renal proximal tubule was also observed in equivalent vesicles prepared from rat small intestine.     

Adherence of Campylobacter jejuni and Campylobacter coli to porcine intestinal brush border membranes

The adhesion of Campylobacter jejuni and C. coli to isolated porcine intestinal brush border membranes was studied by phase-contrast and electron microscopy. Approximately 45% of the cell population adhered to the brush borders, possibly in a specific manner. Pretreatment of the brush borders with trypsin or pronase, and competitive inhibition with L-rhamnose caused a slight reduction of the adhesion. Different forms of pretreatment of the bacterial cells reduced their ability to adhere, but also their motility.     

Sialic acid deficiency in lectin-resistant intestinal brush border membranes from rats following the intestinal phase of trichinellosis

Maximal binding (Bmax) of the lectin, wheat germ agglutinin, by small intestinal brush border membrane is significantly reduced in rats infected with Trichinella spiralis. Wheat germ agglutinin specificity is for N-acetylglucosamine and sialic acid. Whereas total hexosamine and N-acetylglucosaminidase-labile N-acetylglucosamine were comparable in membranes from uninfected as compared with infected rats, the total sialic acid content and neuraminidase-released sialic acid were significantly higher in BBM from uninfected hosts. N-Acetylglucosaminidase treatment of membranes reduced Bmax for wheat germ agglutinin in both hosts. Neuraminidase treatment reduced Bmax in uninfected hosts, but tended to increase it in infected rats. Membranes from uninfected rats incorporated more N-acetylglucosamine from UDP-N-[14C]acetylglucosamine into oligosaccharide-lipid than did membranes from infected hosts. However, lipid and protein fractions were labeled at the same rate in both sets of membranes. Sialic acid was incorporated into protein at a slightly faster rate in brush border membrane from uninfected rats, indicative of a higher level of sialotransferase activity. These results suggest that the reduction in Bmax for wheat germ agglutinin in gut epithelial cell membranes from infected rats is related to a reduced level of sialic acid available for lectin binding as well as specific interactions between N-acetylglucosamine and sialic acid.     

Zinc inhibition of glucose uptake in brush border membrane vesicles from pig small intestine

The effect of zinc on sodium coupled glucose uptake was studied in pig intestinal brush border membrane vesicles. In this system zinc inhibited glucose uptake and appeared to have a K _i of 0.25 mM. When tested by spectrophotometry, electron microscopy and protein determination following centrifugation, no evidence of significant vesicle aggregation was found with 0.5 mM zinc treatment. Zinc inhibition of glucose uptake persisted when the vesicle membrane potential was clamped with identical KCl concentrations inside and outside the vesicles in the presence of valinomycin. Variation of the glucose and sodium concentrations gave results indicating that zinc reduces glucose affinity for the carrier but not sodium binding to the transporter. The glucose inhibitory effect was not due to a rapid dissipation of the sodium gradient as zinc failed to affect sodium uptake in the absence of glucose. Zinc also failed to inhibit glucose efflux from vesicles under isotopic exchange conditions, when glucose and sodium concentrations were identical inside and outside vesicles. The t1/2 of glucose inhibition by zinc was relatively long, i.e. 6 min. We conclude that zinc acts as an inhibitor of glucose transport by interacting with the sodium-glucose co-transporter. The long zinc incubation time required to achieve maximal inhibition of glucose transport suggests that this interaction takes place within vesicles.     

Phosphatidylethanolamine methylation in intestinal brush border membranes from rats resistant to Trichinella spiralis

Methylation of phospholipids is proposed as a mechanism to explain changes in properties of intestinal brush border membrane that coincide with development of immunity to the intraepithelial parasite, Trichinella spiralis. Methylation was measured by the incorporation of the [3H]methyl group from S-adenosyl-L-[3H]methyl methionine into phospholipids. At least two enzymatic components were detected that converted phosphatidylethanolamine to phosphatidylcholine. The first, designated methyltransferase I, catalyzed the formation of phosphatidylmonomethylethanolamine from phosphatidylethanolamine and had a low Km for S-adenosyl-L-methyl-methionine (5 microM). The second, designated methyltransferase II, which catalyzed the methylation of phosphatidylmonomethylethanolamine to phosphatidyldimethylethanolamine and phosphatidyldimethylethanolamine to phosphatidylcholine, had a high Km for S-adenosyl-L-methyl methionine (167 microM). Both enzymes had two pH optima, were most active at 37 degrees C and were Mg2+ dependent. A decrease in methylation activity was present in brush border membranes from rats immunized against T. spiralis. Although the synthesis of phosphatidylcholine was not significantly altered there was a substantial decrease in the formation of phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine as compared with nonimmunized rats. Since phospholipid composition influences membrane fluidity and cell function, it is proposed that altered methylation activity may influence the characteristics of brush border membrane in the immune host.     

Interaction of Escherichia coli K88 antigen with porcine intestinal brush border membranes.

The fimbria-associated Escherichia coli antigen, K88, was purified to homogeneity as determined by polyacrylamide gel electrophoresis and immunodiffusion. This polymeric antigen consists of noncovalently linked subunits, containing little or no carbohydrate, and has a monomeric molecular weight of 23,000. When a binding assay employing differential filtration was used, K88 formed complexes with isolated porcine intestinal brush border membranes. The formation of complexes was inhibited by glycoproteins with terminal N-acetylglucosamine and N-acetylgalactosamine residues and to a lesser extent by free N-acetylhexosamines. These amino sugars may play a role in the interaction of this pathogenic strain of E. coli with the intestinal epithelia of pigs.     

Binding of Escherichia coli heat-stable enterotoxin to rat intestinal cells and brush border membranes

The association of heat-stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli 431 with isolated rat intestinal epithelial cells and brush border membranes was characterized. Specific binding of strain 431 125I-STa to a single class of specific high-affinity receptors was saturable and temperature dependent and reached a maximum between 5 and 10 min. A 1,000-fold excess of unlabeled 431 STa competitively displaced 90 to 95% of radiolabeled enterotoxin bound to brush border membranes. In contrast, specific binding of 431 125I-STa to intestinal cells ranged from 40 to 65%. The number of STa-specific receptors on rat intestinal cells determined by Scatchard analysis was 47,520 +/- 14,352 (mean +/- standard error of the mean) per cell, with affinity constants (KaS) of 2.55 X 10(11)and 4.32 x 10(11) liters/mol determined for intestinal cells and brush border membranes, respectively. Villus intestinal cells appeared to possess about twice as many STa receptors as did crypt cells. Dissociation of specifically bound 431 125I-STa from intestinal cells and brush border membranes was minimal (2 to 5%). In addition, neither the rate nor the extent of dissociation was increased by a 1,000-fold excess of unlabeled homologous 431 Sta. Binding experiments with 431 125I-STa and brush border membranes showed that purified unlabeled STas from enterotoxigenic E. coli strains 667 (class 1 porcine enteropathogen), B-41 (bovine enteropathogen), and human strains 213C2 (Mexico) and 153961-2 (Dacca, Bangledesh) exhibited patterns of competitive inhibition similar to those of homologous unlabeled 431 STa (class 2 enteropathogen). A lipid extract which contained gangliosides and glycolipids exhibited dose-dependent competitive inhibition of heat-labile enterotoxin binding to brush border membranes but did not inhibit binding of 431 125I-STa. Purified heat-labile enterotoxin from strain 286C2 did not inhibit binding of 431 STa to brush border membranes. Pronase treatment of brush border membranes reduced binding of 431 125I-STa by about 30%, suggesting that the STa receptor was a protein or a glycoprotein. The putative STa receptor was radiolabeled with 431 125I-STa and solubilized with sodium deoxycholate. One major radioactive band was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by radioautography. These data suggested that STas bind essentially irreversibly to a specific receptor on the cell surface of intestinal cells before activation of guanylate cyclase.     

The distribution and mobility of anionic sites on the brush border of intestinal absorptive cells

The distribution and behavior of anionic sites on the micro-villous surface of rat jejunal absorptive cells were studied using the multivalent ligand polycationic ferritin (PCF) as a visual probe. Segments were incubated in PCF either before or after glutaraldehyde fixation. The results indicated that the anionic sites can be divided into three groups based on their interaction with PCF: (1) Some sites along the length of the microvilli are accessible for binding PCF in living, unfixed cells. These sites are capable of translational mobility at the membrane surface and can be induced to cluster into discrete patches by PCF. Under appropriate conditions, they may be taken into the cell by endocytosis. Their redistribution is prevented by prefixation, and slowed by incubation at temperatures of 0°C or below. (2) Some sites along the length of the microvilli are inaccessible to PCF without prior fixation. These sites may provide uniform coverage or may be preferentially localized to the distal microvillus. (3) Some sites restricted to the microvillous tips are accessible to PCF without fixation, but are apparently immobile. Their high density on some tips forms a distinctive cap of ferritin particles. More anionic sites were found on the microvilli of the more mature cells of the upper villus. In addition, independent variation was observed in the number of sites in each of the three groups among neighboring cells, irrespective of villous position, suggestive of variations in the turnover of these sites.     

Three antibodies to human intestinal brush border membrane are characterized as anti-Le(a)

In the course of raising monoclonal antibodies to human colonic and small intestinal epithelium, three were isolated which recognized epitopes on many colonic glycoproteins and many jejunal glycoproteins taken from some individuals, but not on the same glycoproteins from others. The specificities of these antibodies were examined by carrying out inhibitions of the binding of the antibodies to the jejunal glycoproteins by blood group substances and purified oligosaccharides. The glycoproteins were separated by electrophoresis and transferred to nitrocellulose strips which were then used for the inhibition experiments. The results indicated that the antibodies bind to Le(a)-active structures. The conditions were determined for achieving red cell agglutination by two of the antibodies.