Preparation Of Brush Border Membrane Vesicles Research Articles

Differential proteome analysis along jejunal crypt-villus axis in piglets

The brush-border membrane (BBM) of enterocytes is responsible for the digestion and absorption of nutrients and ions in the small intestine. To identify the BBM proteins involved in epithelial cell maturation along the crypt-villus axis, enterocytes were sequentially isolated from the villus tip to the crypt of the jejunum from 21-day-old suckling piglets. After preparation of BBM vesicles, we detected 194 proteins in the jejunal epithelial cells by isobaric tags using relative and absolute quantification (iTRAQ) techniques. Of these, 56 BBM proteins were differentially expressed along the crypt-villus axis. During differentiation, the expression of proteins related to digestion and absorption of nutrients was primarily downregulated at the upper, middle villus, or crypt compared to the villus tip, while expression of proteins related to structural and enzyme regulator proteins was largely upregulated. We verified the differences in Na(+)/K(+) -transporting ATPase, galectin-3, and an intestinal-type fatty acid binding protein by western blot or immunochemical analysis. Identification of BBM-associated proteins helps enhance our understanding of digestion and absorption in piglets and other mammals, including humans.

Alterations in the proteome of the NHERF2 knockout mouse jejunal brush border membrane vesicles

To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.

Alterations in the proteome of the NHERF1 knockout mouse jejunal brush border membrane vesicles

Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCβ3, E-cadherin, p120, β-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.

In Vitro Intestinal Absorption of Carotenoids Delivered as Molecular Inclusion Complexes with β-Cyclodextrin Is Not Inhibited by High-Density Lipoproteins

This study analyzed the assimilation efficiency of carotenoids when they are delivered as inclusion complexes with beta-cyclodextrin (CyDIC) in water. The in vitro assimilation model used was the brush border membrane vesicles (BBMV) system in which the BBMVs were incubated with CyDIC. Carotenoid suspensions in Tween were used as a reference. Regardless of the form in which the carotenoids were delivered to the BBMV preparation, a higher assimilation efficiency was observed for carotenes than for the xanthophyll lutein. At the highest donor solution concentration, supplying carotenoids in CyDIC produced a significant increase in carotenoid assimilation compared to the corresponding carotenoid suspensions in Tween. The assimilation process using CyDIC takes place by means of a dissociation process in which the carotenoids are released from the beta-cyclodextrin to later be assimilated. At the highest concentration of CyDIC in the donor solution, the dissociation equilibrium will be shifted toward the free forms of the complex, thus increasing the amount of carotenoids available for assimilation. In another set of experiments, the effect of high-density lipoproteins as activity inhibitors for the receptors involved in carotenoid assimilation was analyzed. In carotenoid suspensions in Tween, with an inhibitor, a significant decrease in the assimilated quantity compared was observed with values reached without the inhibitor. Lutein presented the most significant decrease (70%). The fact that complete inhibition was not reached suggests that both simple and facilitated diffusion contributes to the assimilation process. When the donor solution composed of CyDIC and inhibitor was added, significant increases were observed in beta-carotene and lycopene assimilation for all concentrations and in lutein for the highest concentration. This effect is due to the exchange between lipoprotein lipid components and CyDIC, which promotes the dissociation and liberation processes of the carotenoid, which then becomes available for assimilation.

Aquaporin-6 is expressed along the rat gastrointestinal tract and upregulated by feeding in the small intestine

BackgroundSeveral aquaporins (a family of integral membrane proteins) have been recently identified in the mammalian gastrointestinal tract, and their involvement in the movement of fluid and small solutes has been suggested. In this direction we investigated, in some regions of the rat gastrointestinal tract, the presence and localization of aquaporin-6, given its peculiar function as an ion selective channel.ResultsRT-PCR and immunoblotting experiments showed that aquaporin-6 was expressed in all the investigated portions of the rat gastrointestinal tract. The RT-PCR experiments showed that aquaporin-6 transcript was highly expressed in small intestine and rectum, and less in stomach, caecum and colon. In addition, jejunal mRNA expression was specifically stimulated by feeding.Immunoblotting analysis showed a major band with a molecular weight of about 55 kDa corresponding to the aquaporin-6 protein dimer; this band was stronger in the stomach and large intestine than in the small intestine. Immunoblotting analysis of brush border membrane vesicle preparations showed an intense signal for aquaporin-6 protein.The results of in situ hybridization experiments demonstrate that aquaporin-6 transcript is present in the isthmus, neck and basal regions of the stomach lining, and throughout the crypt-villus axis in both small and large intestine. In the latter regions, immunohistochemistry revealed strong aquaporin-6 labelling in the apical membrane of the surface epithelial cells, while weak or no labelling was observed in the crypt cells. In the stomach, an intense staining was observed in mucous neck cells and lower signal in principal cells and some parietal cells.ConclusionThe results indicate that aquaporin-6 is distributed throughout the gastrointestinal tract. Aquaporin-6 localization at the apical pole of the superficial epithelial cells and its upregulation by feeding suggest that it may be involved in movements of water and anions through the epithelium of the villi.

Class B type I scavenger receptor is responsible for the high affinity cholesterol binding activity of intestinal brush border membrane vesicles

Recent studies have documented the importance of Niemann–Pick C1-like 1 protein (NPC1L1), a putative physiological target of the drug ezetimibe, in mediating intestinal cholesterol absorption. However, whether NPC1L1 is the high affinity cholesterol binding protein on intestinal brush border membranes is still controversial. In this study, brush border membrane vesicles (BBMV) from wild type and NPC1L1 −/− mice were isolated and assayed for micellar cholesterol binding in the presence or absence of ezetimibe. Results confirmed the loss of the high affinity component of cholesterol binding when wild type BBMV preparations were incubated with antiserum against the class B type 1 scavenger receptor (SR-BI) in the reaction mixture similar to previous studies. Subsequently, second order binding of cholesterol was observed with BBMV from wild type and NPC1L1 −/− mice. The inclusion of ezetimibe in these in vitro reaction assays resulted in the loss of the high affinity component of cholesterol interaction. Surprisingly, BBMVs from NPC1L1 −/− mice maintained active binding of cholesterol. These results documented that SR-BI, not NPC1L1, is the major protein responsible for the initial high affinity cholesterol ligand binding process in the cholesterol absorption pathway. Additionally, ezetimibe may inhibit BBM cholesterol binding through targets such as SR-BI in addition to its inhibition of NPC1L1.

Ontogenic changes in lactoferrin receptor and DMT1 in mouse small intestine: implications for iron absorption during early lifeThis paper is one of a selection of papers published in this Special Issue, entitled 7th International Conference on Lactoferrin: Structure, Function, and Applications, and has undergone the Journal's usual peer review process.

It has been proposed that lactoferrin receptor (LfR) may be involved in intestinal iron transport during early life. However, it is known that iron homeostasis is regulated by divalent metal transporter 1 (DMT1; Nramp2/DCT1) in the adult small intestine. To address the hypothesis that LfR may play a role as an alternative iron transport pathway during early life, we used immunohistochemistry (IHC) to examine the localization of mouse LfR (mLfR) and DMT1. In addition to studying the localization and abundance of LfR and DMT1 on the apical membrane, intestinal brush-border membrane vesicles (BBMV) were isolated during the first 3 postnatal weeks (postnatal day (PD) 0, 5, 10, and 20). We found that mLfR is expressed in fetal mice as early as gestational days (GD) 13.5, 15.5, and 18.5. A 34 kD band for mLfR was detected at PD 0 through PD 20 in total intestine homogenate. However, mLfR protein did not appear in the BBMV preparations until PD 5 and was highly expressed at PD 10. By IHC, DMT1 protein was minimally observed at PD 0 and PD 5, but by PD 10 DMT1 was predominantly localized in the apical membrane of the maturing intestine. BBMV fractionation revealed 50-120 kD protein bands for DMT1. In these BBMV preparations, the apical-membrane-associated 120 kD band for DMT1 increased in abundance with age. However, in the corresponding total homogenates, only the deglycosylated form of DMT1 (50 kD) was identified. These results indicate that DMT1 is mislocalized during late gestation, minimally expressed during early life, and predominantly expressed in its deglycosylated form until PD 20. The immunolocalization and abundant protein expression of mLfR suggest that accrual of iron from Lf may be the principal iron uptake pathway at this age. In conclusion, our findings support the notion that until the development-dependent expression of DMT1 in the intestine is induced, mLfR may serve as an alternative iron uptake pathway.

Zinc uptake across the apical membrane of freshwater rainbow trout intestine is mediated by high affinity, low affinity, and histidine-facilitated pathways

Zinc is both a vital nutrient and an important toxicant to aquatic biota. In order to understand the interplay between nutrition and toxicity, it will be important to determine the mechanisms and the factors that regulate zinc uptake. The mechanism of apical intestinal Zn(II) uptake in freshwater rainbow trout and its potential modification by the complexing amino acid histidine was investigated using brush-border membrane vesicles (BBMVs). Following characterisation of the BBMV preparation, zinc uptake in the absence of histidine was both time- and concentration-dependent and consisted of two components. A saturable phase of uptake was described by an affinity constant of 57±17 μM and a transport capacity of 1867±296 nmol mg membrane protein −1 min −1. At higher zinc levels (>500 μM) a linear, diffusive component of uptake was evident. Zinc transport was also temperature-dependent, with Q 10 values suggesting zinc uptake was a carrier-mediated process. Zinc uptake by vesicles in the presence of histidine was correlated to a mono-histidine species (Zn(His) +) at all Zn(II) concentrations examined.

Aminopeptidase-N from the Helicoverpa armigera (Hubner) brush border membrane vesicles as a receptor of Bacillus thuringiensis crylac delta-endotoxin.

Brush border membrane vesicles (BBMVs) were prepared from the 2nd instar larvae of Helicoverpa armigera. Binding of the activated Cry1Ac of Bacillus thuringiensis (Bt) toxin was shown by immunoblot. A 120-kDa protein was identified as a receptor for the Cry1Ac type δ-endotoxin. The aminopeptidase-N activity of BBMVs was measured as the hydrolysis of L-leucine p-nitroanilide. The specific activity was 35 units/mg protein. The BBMV preparation also showed low level of alkaline phosphatase activity. Zn++ chelating agents 2,2′-dipyridyl and 1,10-phenanthroline inhibited aminopeptidase activity at 10 mM concentration, indicating the presence of zinc-dependent aminopeptidase in the brush border of H. armigera. The aminopeptidase activity was increased with increasing concentration of δ-endotoxin. The purified 120-kDa binding protein was N-terminally sequenced. The first 10-amino-acid sequence showed 60–77% similarity with human cysteine-rich secretory protein-1 precursor, inhibin alpha chain precursor. Salmonella flagellar hook protein and yeast carboxypeptidase S.

A Novel Method of Preparation of Small Intestinal Brush Border Membrane Vesicles by Polyethylene Glycol Precipitation

A novel method of brush border membrane vesicle (BBMV) preparation from the small intestinal mucosa using polyethylene glycol (PEG) precipitation has been presented. This preparation is compared with calcium-precipitated BBMVs in marker enzyme enrichment, contamination by other subcellular membranes, transport of glucose, and lipid composition. PEG-precipitated BBMVs are comparable with calcium-precipitated membranes in all parameters except lipid composition and thiol content. PEG-precipitated membranes have more phosphatidylcholine and phosphatidylethanolamine and less lysophosphatidylcholine and lysophosphatidylethanolamine as compared to calcium-precipitated membranes. Diacylglycerol and triacylglycerol content are also high in PEG-precipitatedmembranes. Alteration in lipid composition indicate the possible activation of lipase and phospholipase by calcium during BBMV preparation, which is not seen in PEG precipitation. Thiol content is almost double in PEG-precipitated membranes as compared to calcium-precipitated membranes. These results indicate that PEG can be used for the preparation of BBMVs in native form from the intestine without any alteration in their structural components, and these membranes show comparable transport activity.

Influence of alpha-linked glucose on sodium-glucose cotransport activity along the small intestine in cattle.

Thirteen steers (378+/-23 kg) were used in a split-plot experimental design to evaluate the effect of small intestinal carbohydrate on sodium-glucose cotransport in brush border membrane vesicles prepared from five equidistant sites along the small intestine. The steers consumed 7.2+/-0.4 kg/d ground fescue hay and soybean meal-based supplement and were infused ruminally or postruminally with a partial alpha-amylase starch hydrolysate (914.5+/-8.3 g/d) for 7 d. On d 7, five equidistant 1-m small intestinal sections were harvested and frozen in liquid N for later preparation of brush-border membrane vesicles. Maltase activity of the homogenate and vesicle preparations changed (P < 0.001; lowest in the duodenum, highest in the jejunum) and alkaline phosphatase decreased (P < 0.001) along the small intestine. With respect to the original homogenates, the vesicle preparations were enriched 9.80+/-0.83- and 7.64+/-0.67-fold for alkaline phosphatase and maltase, respectively; enrichments were not different between treatments (P = 0.76 and 0.39, respectively). However, alkaline phosphatase and maltase enrichment changed (P < 0.001) along the small intestine. Recoveries of alkaline phosphatase and maltase activities (25.0+/-0.2% and 19.5+/-0.2%, respectively) in the vesicle preparation were not affected (P = 0.29 and 0.21, respectively) by treatment but changed (P < 0.001) along the intestine. Recovery of protein in the vesicle preparation was 2.60+/-0.01% and was not affected by treatment or intestinal site. Sodium-glucose cotransport activity (220+/-44 pmol x mg(-1) x s(-1)) was not affected (P = 0.34) by treatment but did change (P < 0.001; lowest in the ileum, highest in the proximal and mid-jejunum) along the small intestine. Apparent Km of the sodium-glucose cotransporter for glucose was 62.8+/-5.8 microM. The specific activity of maltase was highest in the jejunum, and sodium-glucose cotransport was highest in the first two jejunal sites. However, duodenal maltase activity was lowest and ileal sodium-glucose cotransport activity was lowest. Sodium-glucose cotransport activity may limit small intestinal starch assimilation in the distal small intestine. It does not seem that glucose arising from carbohydrate hydrolysis regulates activity of sodium-dependent glucose transport in cattle.

Escherichia coli K88 receptor expression in intestine of disease-susceptible weaned pigs

A challenge trial was carried out in which Escherichia coli O157 K88ac was administered to a litter of weaned pigs and the development of the disease monitored over a five-day experimental period. The eight animals in the trial were assigned to two groups depending on whether they exhibited disease symptoms. Six pigs developed diarrhoea and two appeared unaffected; these were designated as the test (or K88-susceptible) group and the control (or K88-resistant) group, respectively. The animals were euthanised and the intestine was removed and sections processed for brush border membrane vesicle preparation. Microscopic and biochemical assays were undertaken on tissue samples from each animal and a strong correlation was observed between the expression of a glycoprotein receptor complex associated with the brush border membrane and the development of disease symptoms. Further investigation revealed the presence of an analogous glycoprotein complex in the K88-resistant group which did not bind the K88-fimbriae antigen. These results suggest that genetic differences in the glycosyl moieties of the receptor complex provide the basis for disease susceptibility to K88-positive E. coli.

Bacillus thuringiensis HD-73 Spores Have Surface-Localized Cry1Ac Toxin: Physiological and Pathogenic Consequences

Spores from Cry(sup+) strains of Bacillus thuringiensis bound fluorescein isothiocyanate-labeled antibodies specific for the 65-kDa activated Cry 1Ac toxin, whereas spores from Bacillus cereus and Cry(sup-) strains of B. thuringiensis did not. The Cry(sup+) spores could be activated for germination by alkaline conditions (pH 10.3), whereas Cry(sup-) spores could not. Once the surrounding exosporia had been removed or permeabilized, Cry(sup+) spores were able to bind the toxin receptor(s) from insect gut brush border membrane vesicle preparations, and their germination rates were increased ca. threefold in the presence of brush border membrane vesicles. A model is presented whereby in the soil the Cry toxins on the spore surface are protected by the exosporium while in the gut they are exposed and available for binding to the insect receptors. This model explains why the disulfide-rich C terminus of the cry genes is so highly conserved even though it is removed during the processing of the protoxin to the activated toxin. It also highlights the trade-off resulting from having Cry toxins located on the spore surface, i.e., decreased spore resistance versus enhanced insect pathogenesis.

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

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

Substrate-charge dependence of stoichiometry shows membrane potential is the driving force for proton-peptide cotransport in rat renal cortex.

The proton dependence of the transport of three labelled, hydrolysis-resistant synthetic dipeptides carrying a net charge of -1, 0 or +1 has been investigated in a brush border membrane vesicle preparation obtained from rat renal cortex. Cross-inhibition studies are consistent with the transport of all peptides studied being through a single system. The extent and time course of uptake in response to an inwardly directed electrochemical gradient of protons differed for each peptide. For the cationic peptide D-Phe-L-Lys this gradient did not stimulate the initial rate of uptake, while for the neutral dipeptide D-Phe-L-Ala and the anionic peptide D-Phe-L-Glu stimulation was observed. However, the effect on D-Phe-L-Glu was more marked than that on D-Phe-L-Ala and the proton activation differed for these two peptides. The calculated Hill coefficients for the two proton-dependent peptides were 1.14 +/- 0.16 and 2.15 +/- 0.10 for D-Phe-L-Ala and D-Phe-L-Glu, respectively, providing evidence that the stoichiometry of proton:peptide cotransport is different for each peptide (0:1, 1:1 and 2:1 for D-Phe-L-Lys, D-Phe-L-Ala and D-Phe-L-Glu respectively); studies on energetics are compatible with this conclusion. The physiological and molecular implications of this model are discussed, as are the applicability of the conclusions to secondary active transport systems more generally.

Cadmium-induced nephrotoxicity in rhesus monkeys ( Macaca, mulatta) in relation to protein calorie malnutrition

In this study, we compared results obtained in protein calorie malnourished (PCM) monkeys and controls given Cd 2+ (5 mg Cd 2+/kg body wt./day) orally for 24 weeks. After 16 weeks of Cd 2+ exposure, an indolent renal failure develops in PCM monkeys which resulted in significant increase in urinary excretion of total protein, Cd 2+, Zn 2+ and Ca 2+ as compared to corresponding Cd 2+-treated control group. In isolated proximal tubule brush border membrane vesicles (BBMV), Cd 2+, Zn 2+ and Ca 2+ transport were significantly impaired in Cd 2+-exposed PCM monkeys as compared to Cd 2+-treated controls. The mechanism of higher urinary excretion of Cd 2+, Zn 2+ and Ca 2+ was examined by analyzing the kinetic parameters of transport systems. The kinetic studies of Cd 2+, Zn 2+ and Ca 2+ transport systems in the BBMV preparations of Cd 2+-exposed PCM monkeys exhibited a significant decrease in V max and an appreciable increase in K m as compared to Cd 2+-treated controls. These findings suggested that Cd 2+ treatment of PCM monkeys caused either a decrease in the number of transporters in the brush border membrane or an increase in the number of less active transporters for Cd 2+, Zn 2+ and Ca 2+. Furthermore, brush border membrane-bound enzymes, viz. alkaline phosphatase and leucine aminopeptidase, activities were significantly impaired in Cd 2+-exposed PCM monkeys. Cadmium content in kidney cortex of Cd 2+-exposed PCM monkeys was 3.34-fold higher than Cd 2+-exposed controls. These findings also established that Cd 2+ not bound to metallothionein (MT) was significantly higher in Cd-exposed PCM monkeys, which may be an important determinant in renal toxicity by interacting with sensitive sites in the renal cells and causing renal damage in Cd-exposed PCM monkeys.

Effect of high osmolality on Na+/H+ exchange in renal proximal tubule cells.

Na+/H+ exchanger isoform and the effect of high osmolality on its function was studied in cultured renal epithelial cells (LLC-PK1 and OK). Using NHE-3-specific antibody, immunoblots of luminal membranes from LLC-PK1 and OK cells specifically labeled proteins with molecular masses 90 and 95 kDa, indicating that NHE-3 is the isoform expressed on the luminal membranes of these epithelia. Proximal tubular suspensions from rabbit kidney cortex were incubated in control (310 mosm/liter) or high osmolality (510 mosm/liter) medium for 45 min and utilized for brush border membrane vesicle preparation. Influx of amiloride-sensitive 22Na+ at 10 s (pHo 7.5, pHi 6.0) into brush border membrane vesicles was 37% lower in the high osmolality group (p < 0.03). LLC-PK1 or OK cells were grown to confluence and examined for Na+/H+ exchange activity. An increase in medium osmolality to 510 mosm following acid loading decreased the 5-min uptake of the amiloride-sensitive 22Na+ in LLC-PK1 and OK cells (p < 0.04 and < 0.03 for LLC-PK1 cell OK cells, respectively). An increase in medium osmolality to 510 mosm in vascular smooth muscle cells, which express NHE-1, produced 45 and 64% stimulation of the amiloride-sensitive 22Na+ influx at base-line pHi and acid-loaded condition, respectively (p < 0.03 and < 0.01). Down-regulation of protein kinase C by preincubation with phorbol 12-myristate 13-acetate or inhibition of Ca(2+)-calmodulin-dependent protein kinase (calmodulin-kinase II) by N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide (W-7) in LLC-PK1 cells did not block the inhibitory effect of high osmolality on Na+/H+ exchange activity. We conclude that renal proximal tubule epithelial cells express Na+/H+ exchange isoform NHE-3 on their luminal membranes and that hyperosmolality decreases transporter activity during cell acidification. This inhibitory effect might be unique to the NHE-3 isoform, since vascular smooth muscle cells which express NHE-1 exhibit an increase in Na+/H+ exchange activity in response to high osmolality.

Uptake of riboflavin across the brush border membrane of rat intestine: Regulation by dietary vitamin levels

Background : Possible regulation of riboflavin (RF) intestinal uptake process by dietary substrate level is not known and was examined in this study. Methods : RF uptake was examined using intestinal brush border membrane vesicles (BBMV). Results : Oversupplementation with RF caused significant decrease ( P < 0.05; down-regulation) in the uptake of 3 μmol/L RF compared with control. This effect was not due to differences in the relative purity of the intestinal BBMV preparations and appeared to be specific for RF. This down-regulation was mediated through a significant ( P < 0.01) decrease in the V max of the RF uptake process with no significant change in the apparent Michaelis constant ( K m ). In contrast, RF deficiency caused a significant ( P < 0.01) enhancement (up-regulation) in the uptake of 3 μmol/L RF compared with pair-fed control. Again the enhancement was not due to differences in the relative purity of the BBMV preparations and appeared to be specific for RF. This up-regulation was mediated via a significant ( P < 0.01) increase in the V max of the RF uptake process with no significant change in the apparent K m . Conclusions : These findings show that the RF intestinal uptake process is regulated by the level of the vitamin in the diet (and/or body stores) and that the regulation is mediated via changes in the number (and/or activity) of the RF uptake carriers with no change in their affinity.

Peptide carrier-mediated transport in intestinal brush border membrane vesicles of rats and rabbits: cephradine uptake and inhibition.

The uptake kinetics of cephradine, an amino-beta-lactam antibiotic, were studied in rat and rabbit intestinal brush border membrane vesicles preparations using both the Ca2+ and the Mg2+ methods of preparation, in the presence of an inward proton gradient. The Ca2+ method demonstrated greater uptake of cephradine in intestinal brush border vesicles prepared from both rat and rabbit and was used for these studies. The transport was observed to be of Michaelis-Menten carrier-mediated type with a passive transport component. The kinetic parameters obtained were as follows: for rat and rabbit, respectively, Km, 1.6 and 1.9 mM; Jmax', 1.7 and 20.7 nmol/mg/min; Pc' (= Jmax'/Km), 1.1 and 10.9 microL/mg/min; and Pm', 0.4 and 0.8 microL/mg/min. The kinetic parameters for the rat vesicles are consistent with those from our previous perfusion study using a conversion factor of 0.71 cm2/mg protein. The rabbit vesicles exhibited a similar Michaelis constant and a 10-fold larger maximal transport velocity, suggesting a quantitative advantage for the study of carrier-mediated transport in the rabbit compared to rat vesicles from the intestine. Cephradine uptake was inhibited by phenylpropionylproline, a proline derivative, and enalapril, an ACE inhibitor, which do not have an alpha-amino group, as well as dipeptides, tripeptides, and amino-beta-lactam antibiotics in both rat and rabbit vesicles. These results support the suggestion that they share the same peptide carrier pathway for oral absorption and that the vesicles may be a useful tool in developing orally effective peptide-type drugs.

Efficiency of amino acid transport into preserved brush border membrane vesicles from lepidopteran larval midgut

1. 1. The effect of different conditions of preservation of the larval midgut tissue of Bombyx mori and Philosamia cynthia on the efficiency of amino acid transport into brush border membrane vesicles (BBMV) has been studied. 2. 2. Freezing and storage of the tissue in liquid nitrogen followed by rapid thawing at 37°C allowed the preparation of BBMV which retained fully for B. mori and partially for P. cynthia the ability to perform l-histidine transport and accumulation. 3. 3. BBMV prepared from fresh midguts of B. mori, immediately frozen and preserved in liquid nitrogen for different periods, lost part of their transport efficiency at the moment of freezing, independently of the time of storage. 4. 4. A decrease was also observed for preserved BBMV from P. cynthia midgut. However, the addition of a cryoprotectant such as glycerol to BBMV improved the performance of the reconstituted preparation, 5. 5. The transport capacity of the stored vesicles depended also on their slow thawing on ice.