Thyroid hormones increase Na+–Pi co-transport activity in intestinal brush border membrane: Role of membrane lipid composition and fluidity

In the present study, we documented the promising role of thyroid hormones status in animals in modulation of Na⁺–P_i transport activity in intestinal brush border membrane vesicles (BBMV) which was accompanied with alterations in BBM lipid composition and fluidity. Augmentation of net P_i balance in hyperthyroid (Hyper-T) rats was fraternized with accretion of P_i transport across BBMV isolated from intestine of Hyper-T rats as compared to hypothyroid (Hypo-T) and euthyroid (Eu-T) rats while Na⁺–P_i transport across BBMV was decreased in Hypo-T rats relative to Eu-T rats. Increment in Na⁺–P_i transport in intestinal BBMV isolated from Hyper-T rats was manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport system. Furthermore, BBMV lipid composition profile in intestinal BBM from Hyper-T was altered to that of Hypo-T rats and Eu-T rats. The molar ratio of cholesterol/phospholipids was higher in intestinal BBM from Hypo-T rats. Fluorescence anistropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the intestinal BBM of Hyper-T rats and decreased in Hypo-T rats as compared to Eu-T rats which corroborated with the alteration in membrane fluidity in response to thyroid hormone status of animals. Therefore, thyroid hormone mediated change in membrane fluidity might play an important role in modulating Na⁺–P_i transport activity of intestinal BBM. (Mol Cell Biochem 278: 195–202, 2005)     

Multiple plasma membrane receptors but not NPC1L1 mediate high-affinity, ezetimibe-sensitive cholesterol uptake into the intestinal brush border membrane

We compared cholesterol uptake into brush border membrane vesicles (BBMV) made from the small intestines of either wild-type or Niemann-Pick C1-like 1 (NPC1L1) knockout mice to elucidate the contribution of NPC1L1 to facilitated uptake; this uptake involves cholesterol transport from lipid donor particles into the BBM of enterocytes. The lack of NPC1L1 in the BBM of the knockout mice had no effect on the rate of cholesterol uptake. It follows that NPC1L1 cannot be the putative high-affinity, ezetimibe-sensitive cholesterol transporter in the brush border membrane (BBM) as has been proposed by others. The following findings substantiate this conclusion: (I) NPC1L1 is not a brush border membrane protein but very likely localized to intracellular membranes; (II) the cholesterol absorption inhibitor ezetimibe and its analogues reduce cholesterol uptake to the same extent in wild-type and NPC1L1 knockout mouse BBMV. These findings indicate that the prevailing belief that NPC1L1 facilitates intestinal cholesterol uptake into the BBM and its interaction with ezetimibe is responsible for the inhibition of this process can no longer be sustained.     

Intestinal mucosal lipid peroxidation and absorptive function In Salmonella typhimurium mediated intestinal infection

S. typhimurium infection is associated with neutrophil infiltration within the intestinal mucosa. Neutrophil activation provides a major source of reactive oxygen species (ROS). The mucosal pathology of S. typhimurium infection may be in part due to the excessive production of these reactive species. This study was carried out to investigate if ROS play a role in mediating the changes in the structural components and functional properties of brush border membrane (BBM) in rats during S. typhimurium infection. This was done by determining the changes in the BBM extent of lipid peroxidation and absorptive function. A significant increase in the extent of lipid peroxidation of BBM during S. typhimurium infection was observed as judged by malondialdehyde (MDA) and conjugated diene formation and depletion of -tocopherol and protein associated thiol groups. A significant decrease in the BBMV (brush border membrane vesicle) transport of amino acids was also observed. However there was no change in the transport of D-glucose. The decrease in amino acid transport further led to a significant decrease in the enterocyte level of protein synthesis. Exposure of BBMV to a free radical donor, cumene hydroperoxide, also led to an increase in the extent of lipid peroxidation and a decrease in the amino acid transport. Possibly ROS might play a significant role in mediating the mucosal damage during S. typhimurium infection.     

Effect of cisplatin on brush border membrane enzymes and anti-oxidant system of rat intestine

Cisplatin (CP) is a widely used antineoplastic agent which exhibits gastrointestinal toxicity. The present work was done to study the effect of administration of CP on brush border membrane (BBM) enzymes and anti-oxidant system of rat intestine. Male Wistar rats were given a single intraperitoneal dose of CP (6 mg/kg body weight) and then sacrificed 1, 3, 5 and 7 days after this treatment. Control animals were given saline only. The administration of CP led to significant decline in the specific activities of BBM enzymes both in the mucosal homogenates and isolated membrane vesicles. Kinetic studies showed that the V(max) of the enzymes was decreased in BBM vesicles from CP treated rats while the K(m) remained unchanged. The activities of catalase, Cu-Zn superoxide dismutase, glucose 6-phosphate dehydrogenase and glutathione reductase decreased while the activities of glutathione S-transferase and thioredoxin reductase increased in CP treated animals compared to the control group. Lipid peroxidation and total sulfhydryl groups were also altered upon CP treatment indicating the generation of oxidative stress. The maximum changes in all the parameters studied above were 3 days after administration of CP and then recovery took place on days 5 and 7. Thus, the administration of CP leads to significant alterations in the activities of BBM enzymes and the anti-oxidant status of rat intestine.     

Spontaneous cholesterol movement between lipid vesicles and monkey small intestinal brush border membrane

[14C]Cholesterol movement between egg phosphatidylcholine-cholesterol lipid vesicles and vesicles prepared from monkey small intestinal brush border membrane (BBMV) was studied in physiological buffer at 37 degrees C. The rate of cholesterol transfer from sonicated unilamellar vesicles (ULV) to BBMV follows apparently first-order kinetics. Intermembrane cholesterol movement was strikingly similar in both the directions. However, from BBMV to ULV, the transfer rate was three times faster than that of ULV to brush border membrane (BBM). Similarity in the rate constant was observed when cholesterol transfer was studied using either large multilamellar lipid vesicles or ULV as the donor and BBMV as the acceptor membrane. Rate constant was also the same when the acceptor membrane used was either intact BBMV or ULV prepared from BBM lipids. The rate of transfer of label was not affected even when the acceptor vesicle concentration was increased over fivefold, indicating the first-order nature of the reaction. Transfer of cholesterol from ULV to BBMV was accelerated by the presence of acetone, dimethyl sulfoxide (DMSO), deoxycholate, and papain. Partially purified nonspecific lipid-exchange protein increased the rate of cholesterol transfer by about threefold. Reduction in BBM cholesterol and phospholipid content was noted by DMSO, acetone, and deoxycholate, while papain caused a small depletion of membrane protein. Cholesterol transfer is temperature dependent with an activation energy of 31 kJ X mol-1, which is almost identical in the presence or absence of nonspecific lipid-exchange protein. The molecular mechanism of intermembrane cholesterol movement is discussed in view of the kinetic data obtained.     

Uptake of selenate and selenite by isolated intestinal brush border membrane vesicles from pig,sheep, and rat

Selenate and selenite uptakes by isolated intestinal brush border membrane vesicles (BBMV) from pig, sheep, and rat were investigated. Selenate uptake into jejunal and ileal, but not duodenal, BBMV from pig was stimulated by an inwardly directed transmembrane Na⁺ gradient (Na _out ⁺ >Na _in ⁺ ). Selenate transport into rat ileal and sheep jejunal BBMV was also enhanced in the presence of a Na⁺ gradient. Unlike selenate uptake, selenite uptake was not Na⁺ dependent, neither in pig small intestine nor in sheep jejunum and rat ileum. Uptake of selenate represented real uptake into the vesicular lumen, whereas selenite uptake was a result of an extensive binding of⁷⁵Se to the membranes. Thiosulfate at a 250-fold concentration of selenate completely inhibited Na⁺-dependent selenate uptake into pig jejunal BBMV. Furthermore, Na⁺-dependent sulfate uptake was totally inhibited in the presence of a 250-fold selenate concentration. The results clearly show that selenate transport across the BBM of pig jejunum and ileum, sheep jejunum, and rat ileum is partially energized by a transmembrane Na⁺ gradient. Moreover, it is concluded from the results that there exists a common transport mechanism for sulfate and selenate in the BBM. The extensive binding of⁷⁵Se from⁷⁵Se-labeled selenite to the membranes could be from a spontaneous reaction of selenite with membrane-associated SH groups.     

Comparison of brush border membrane vesicles prepared by three methods from larval Manduca sexta midgut

Brush border membrane vesicles (BBMV) were prepared from freshly isolated posterior larval Manduca sexta midguts by differential calcium precipitation, differential magnesium precipitation and differential ultrasonication. BBMV were also prepared from frozen posterior larval M. sexta midguts by differential calcium precipitaion and differential magnesium precipitation. The yield of BBMV by both differential precipitation methods was 5–6 times greater than that by the differential ultrasonification method. Enrichments of the brush border membrane marker enzymes alkaline phosphatase, γ-glutamyl transferase, and aminopeptidase were similar in all preparations. The polypeptide composition of all preparations was also similar. The specific activity of mitochondrial and microsomal marker enzymes was higher in BBMV prepared from freshly isolated midguts by the differential precipitation methods than in BBMV prepared by the ultrasonication method. The specific activity of cytochrome-c oxidase was 2.5–7 times higher in BBMV prepared from frozen midguts than in BBMV prepared from fresh tissue.     

Regional variations in intestinal brush border membrane fluidity and function during diabetes and the role of oxidative stress and non-enzymatic glycation

The physical state (fluidity) of lipids modulates the activities of several membrane bound enzymes and transport proteins. Alteration of brush border membrane (BBM) fluidity is one of the several changes exhibited by the small intestine during diabetes. In the present study, an investigation of the diabetes induced regional changes in fluidity, oxidative damage, non-enzymatic glycation as well as the activities and the kinetic parameters of the enzymes alkaline phosphatase and -glutamyl transpeptidase was carried out on the intestinal BBM. At the end of 6 weeks of diabetes, significant increases in the extent of both oxidative damage and non-enzymatic glycation were observed along the length of the intestine along with a simultaneous decrease in membrane fluidity. A significant correlation between the decrease in BBM fluidity and increase in non-enzymatic glycation was observed in the duodenum and jejunum. Additionally regional variations in the activities and kinetic parameters of both the enzymes were observed.     

Effect of excretory-secretory products of Giardia lamblia on glucose and phenylalanine transport in the small intestine of Swiss albino mice

The transport of D-glucose and L-phenylalanine was measured in intestinal brush border membrane (BBM) vesicles treated with Excretory-secretory (ES) products of Giardia lamblia. Uptake was found to be significantly lower (P/0.01) in the treated vesicles than in the controls. Exposure of intestinal tissue to ES products resulted in net secretion (P/0.01) of Na+, C1- and 3-O-methyl-D-glucose. Both observations indicate that alterations in the absorptive functions of the intestine might be attributed to interaction of ES products with the BBM.     

Influence of age on duodenal brush border membrane and specific activities of brush border membrane enzymes in Wistar rats.

To examine age-related changes in the morphology of intestinal brush border membrane (BBM; microvilli) and specific activities of intestinal BBM enzymes including alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (gamma-GT), and disacchridase, four groups of Wistar rats were sacrificed at 2.5 wk, 5 wk, 5 mon and 23 mon. In an electron microscopic examination, morphologically a less dense BBM structure in the duodenum of rats aged 23 mon was observed than that of rats aged 5 mon. Specific activity of ALP in the duodenum from 5-mon-old rats was significantly higher than from rats aged 2.5 wk and 23 mon. The mucosal tissues from 5-wk-old rats had significantly higher specific activity of gamma-GT than did tissues from the other ages. In sucrase and maltase specific activities, 5-mon-old rats had higher activities of these enzymes than other age groups, especially 2.5-wk- and 23-mon-old rats. There was also a significant effect of site on intestinal BBM enzyme activities in post-weanling rats. Regional gradients of ALP and gamma-GT along the entire small intestine (duodenum > jejunum > ileum) were remarkable. Disaccharidase activities peaked in the jejunum and declined toward both the duodenum and ileum. Taken together the result obtained here suggested that 5-mon-old rats had the most elevated intestinal function. This result also strongly indicated that the structure of the intestinal BBM and development of intestinal BBM enzymes in Wistar rate were markedly influenced by age during the postnatal period.     

A proteomic approach to study Cry1Ac binding proteins and their alterations in resistant Heliothis virescens larvae

Binding of the Bacillus thuringiensis Cry1Ac toxin to specific receptors in the midgut brush border membrane is required for toxicity. Alteration of these receptors is the most reported mechanism of resistance. We used a proteomic approach to identify Cry1Ac binding proteins from intestinal brush border membrane (BBM) prepared from Heliothis virescens larvae. Cry1Ac binding BBM proteins were detected in 2D blots and identified using peptide mass fingerprinting (PMF) or de novo sequencing. Among other proteins, the membrane bound alkaline phosphatase (HvALP), and a novel phosphatase, were identified as Cry1Ac binding proteins. Reduction of HvALP expression levels correlated directly with resistance to Cry1Ac in the YHD2-B strain of H. virescens. To study additional proteomic alterations in resistant H. virescens larvae, we used two-dimensional differential in-gel electrophoresis (2D-DIGE) to compare three independent resistant strains with a susceptible strain. Our results validate the use of proteomic approaches to identify toxin binding proteins and proteome alterations in resistant insects.     

Purification of brush border membrane vesicles from rat renal cortex by size-exclusion chromatography.

Size-exclusion chromatography with controlled pore glass (CPG) was used in the further purification of renal brush border membrane vesicles (BBMV) isolated by the Ca precipitation method. The BBMV obtained had an almost spherical shape and their average diameter was about 95 nm in isotonic solution. The specific activities of alkaline phosphate and leucine aminopeptidase in the BBMV preparation were increased 18- and 17-fold, respectively, over those in the crude homogenate. The uptake of D-glucose by the purified BBMV in the presence of a sodium gradient reached 8.53 nmol/mg protein at 20 s. These results indicate that CPG chromatography is suitable procedure by which to obtain purified renal BBMV of homogenous size and with high specific marker enzyme activity for use in the study of membrane transport.     

Chronic cold stress-induced alterations in brush border membrane composition and enzyme activities in rat intestine

The effect of chronic cold stress on the composition and function of rat intestinal brush border membrane (BBM) was studied. Various lipid fractions from intestinal BBM viz. cholesterol (p < 0.01), phospholipids (p < 0.01), triglycerides (p < 0.05) and gangliosides (p < 0.05) were significantly reduced in cold stressed animals, as compared to controls. Analysis of membrane saccharide content revealed a significant increase in sialic acid (25%) and hexosamine (36%) contents and a reduction in fucose (19%) content in cold stressed rats. Determination of various enzyme activities in BBM showed significantly enhanced activities of alkaline phosphatase ( p < 0.01), lactase ( p < 0.001) and leucine aminopeptidase ( p < 0.001), whereas sucrase activity was reduced ( p < 0.05) under these conditions. The magnitude and site of these alterations across the crypt-villus axis varied from enzyme to enzyme. These findings suggest that chronic cold stress results in profound alterations in intestinal BBM. Altered structure and function of intestinal BBM may play a role in stress-induced derangements in gastrointestinal tract.     

Selective transport of the mulberry leaf urease from the midgut into the larval hemolymph of the silkworm,Bombyx mori

Just before spinning, larvae of the silkworm, Bombyx mori, absorb intact urease of the host plant (mulberry leaf) from the midgut lumen into the hemolymph. In order to investigate whether the transport of the mulberry leaf urease is selective, crude proteins extracted from the mulberry leaves were labeled with biotin and orally administered to the fifth instar larvae. The biotinylated proteins transported into the hemolymph were detected by ligand blotting using streptavidin. When the biotinylated proteins were administered to 5-day-old fifth instar larvae, a strong signal of a biotinylated protein was detected in the hemolymph 2 days after the administration. In contrast, when the biotinylated mulberry leaf proteins were administered to 3-day-old fifth instar larvae, no signal derived from the biotinylated proteins was detected in the hemolymph. The signal weakened when the biotinylated proteins had been immunoprecipitated before administering to the larvae, indicating that the signal came from the mulberry leaf urease. These results show that the transport of the mulberry leaf urease from the midgut into the hemolymph is selective and larval-stage specific. Subsequently, binding assays were carried out to test the binding ability of the mulberry leaf urease to the brush border membrane in the epithelial cells of larval midgut. The urease was not bound to the brush border membrane vesicles (BBMV) from the midgut of 3-day-old fifth instar larvae, while more than 60% of the total amount of incubated urease was bound to the BBMV from the midgut of 6-day-old fifth instar larvae. The urease binding ability of BBMV correlated with the uptake of the mulberry leaf urease. This suggests that a urease binding molecule(s) exists in the BBM of the midgut epithelium, which is involved in the uptake of the mulberry leaf urease. In addition, the uptake of the mulberry leaf urease into the hemolymph was induced by 20-hydroxyecdysone.     

Proteomics analysis of Trichoplusia ni midgut epithelial cell brush border membrane vesicles

The insect midgut epithelium is composed of columnar, goblet, and regenerative cells. Columnar epithelial cells are the most abundant and have membrane protrusions that form the brush border membrane (BBM) on their apical side. These increase surface area available for the transport of nutrients, but also provide opportunities for interaction with xenobiotics such as pathogens, toxins and host plant allelochemicals. Recent improvements in proteomic and bioinfbrmatics tools provided an opportunity to determine the proteome of the T. ni BBM in unprecedented detail. This study reports the identification of proteins from BBM vesicles (BBMVs) using single dimension polyacrylamide gel elec? trophoresis coupled with multi-dimensional protein identification technology. More than 3000 proteins were associated with the BBMV of which 697 were predicted to possess either a signal peptide, at least one transmembrane domain or a GPI-anchor signal. Of these, bioinfbrmatics analysis and manual curation predicted that 185 may be associated with the BBMV or epithelial cell plasma membrane. These are discussed with respect to their predicted functions, namely digestion, nutrient uptake, cell signaling, development, cell-cell interactions, and other functions. We believe this to be the most detailed proteomic analysis of the lepidopteran midgut epithelium membrane to date, which will provide information to better understand the biochemical, physiological and pathological processes taking place in the larval midgut.     

Transport of divalent transition-metal ions is lost in small-intestinal tissue of b/b Belgrade rats

Belgrade rats exhibit microcytic, hypochromic anemia and systemic iron deficiency due to a glycine-to-arginine mutation at residue 185 in a metal ion transporter of a divalent metal transporter/divalent cation transporter/solute carrier 11 group A member 2 or 3 (DMT1/DCT1/SLC11A2), a member of the natural-resistance-associated macrophage protein (Nramp) family. By use of rabbit duodenal tissue, a calcein fluorescence assay has previously been developed to assess transport of divalent metal ions across the small-intestinal brush border membrane (BBM). The assay was readily applied here to rat BBM to learn if it detects DMT1 activity. The results demonstrate protein-mediated transport across the BBM of all tested ions: Mn(2+), Fe(2+), and Ni(2+). Transport into BBM vesicles (BBMV) from (b/b) Belgrade rats was below the detection limit. BBMV of +/b origin had substantial activity. The kinetic rate constant for Ni(2+) membrane transport for +/b BBMV was within the range for normal rabbit tissue. Vesicles from +/b basolateral membranes (BLM) showed similar activity to BBMV while b/b BLM vesicles (BLMV) lacked transport activity. Immunoblots using isoform-specific antibodies demonstrated that intestinal levels of b/b DMT1 were increased compared to +/b DMT1, reflecting iron deficiency. Immunoblots on BBMV indicated that lack of activity in b/b vesicles was not due to a failure of DMT1 to localize to the BBMV; an excess of specific isoforms was present compared to +/b BBMV or duodenal extracts. Immunoblots from BLMV also exhibited enrichment in DMT1 isoforms, despite their distinct origin. Immunofluorescent staining of thin sections of b/b and +/b proximal intestines confirmed that DMT1 localized similarly in mutant and control enterocytes and showed that DMT1 isoforms have distinct distributions within intestinal tissue.     

Isolation and biochemical characterisation of lipid rafts from Atlantic cod (Gadus morhua) intestinal enterocytes

Lipid rafts are glycosphingolipid/cholesterol-enriched membrane microdomains that have been extensively studied during the past two decades. Our aim was to isolate and perform biochemical characterization of lipid rafts from the intestinal brush border membrane (BBM) of Atlantic cod (Gadus morhua) to confirm their existence in a cold-water species and compare their characteristics with lipid rafts from other species in terms of lipid and protein content. To validate the isolation process, we assayed marker enzymes for subcellular organelles, including alkaline phosphatase (AP) and leucine aminopeptidase (LAP), both well-known marker enzymes for BBM and lipid rafts. All biochemical methods showed enrichment of AP in both the BBM and lipid raft fractions. Proteomic studies were performed by MALDI-TOF mass spectrometry using trypsin digested SDS-PAGE samples. Various proteins were associated with the cod intestinal lipid raft preparation such as aminopeptidase-N, prohibitin, and beta-actin. Lipid analysis with ³¹P NMR and thin layer chromatography on BBMs and lipid rafts samples gave higher content of sphingomyelin than previously reported in the BBM and lower content of phosphatidylcholine. Furthermore, sphingomyelin was highly dominant in the lipid rafts together with cholesterol. The existence of lipid rafts containing previously reported lipid raft characteristics from the cod intestine has, therefore, been confirmed in a ray-finned fish for the first time to the best of our knowledge.     

Dexamethasone blocks adaptive increase of Na+-Pi cotransport in renal brush border membrane elicited by thyroid hormone

Dexamethasone administered to rats blocks and/or reverses adaptive increases in the rate of Na+-Pi cotransport, and also in the Na+-dependent binding of [14C]-phosphonoformic acid (PFA) by renal brush border membrane (BBM) vesicles elicited by thyroid hormone (T3). In contrast, dexamethasone had no effect on Na+-independent binding of [14C]-phosphonoformic acid, on Na+-dependent transport of D-glucose or on Na+-dependent binding of phlorizin by BBMV which indicates that its inhibitory effect is specific for Na+-Pi cotransport system of BBM. These findings suggest that glucocorticoids antagonize T3-elicited adaptive enhancement of Na+-Pi cotransport in renal proximal tubules by blocking the T3-stimulated de novo synthesis of Na+-Pi symporters and/or their insertion into BBM.     

Effects of a low dietary linoleic acid level on intestinal morphology and enterocyte brush border membrane lipid composition.

The influence of low dietary linoleic acid level (an essential fatty acid deficiency) on the intestine mucosal morphology and the purified brush border membrane (BBM) lipid composition was investigated in the rat. Electron micrographs and morphometric measurements showed that villi and crypt sizes as well as the ultrastructure of epithelial cells were altered. Cholesterol (CHOL) and phospholipid (PL) levels, CHOL/PL ratio and PL class distribution were not changed by the low linoleate diet. However, the fatty acid composition of phospholipids was markedly modified in the enterocyte BBM, showing elevated amounts of palmitoleic (16:1n-7), oleic (18:1n-9) and 5,8,11-eicosatrienoic (20:3n-9) acids and, by contrast, depressed linoleic (18:2n-6) and arachidonic (20:4n-6) acid levels. Although the underlying mechanisms remain unknown the results obtained suggest that essential fatty acids (EFA) could be directly involved in the trigger action of the observed alterations, as regards both their dynamic (metabolic) and structural roles.     

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.