Distinct mechanisms of zinc uptake at the apical and basolateral membranes of caco-2 cells.

Zinc uptake mechanisms at the apical and basolateral membrane borders of caco-2 cells were examined. This human-derived cell line possesses many morphological and functional characteristics of absorptive small intestinal cells. By day 14, confluent and well-differentiated monolayers were formed when the cells were grown on porous polycarbonate filters. Labelled zinc was placed on the apical or basal side of the monolayer and its uptake by the cells, as well as its transport across the monolayer, were measured. Zinc uptake by the cells from the apical side was found to be a saturable process (Kt = 41 microM; Vmax = 0.3 nmols/cm2/10 min) with a diffusional term at higher concentrations (1.0 sec/cm). Apical uptake was not affected by metabolic inhibitors or potential zinc ligands. Zinc uptake from the basolateral side was concentration dependent (Kd = 1.3 sec/cm) and was partially inhibited (30%) by ouabain and vanadate, suggesting that the (Na-K)-ATPase on the basolateral membrane is involved in the serosal uptake of zinc by the cell. Transport of zinc across the monolayers from the apical or basolateral compartment was concentration dependent and was not affected by metabolic inhibitors. Zinc transport from the basolateral side was greater than 2-fold greater than apical transport. Hence, separate mechanisms can be distinguished with respect to zinc uptake at the apical and basolateral membranes of caco-2 cells.     

The use of BeWo cells as an in vitro model for placental iron transport

BeWo cells are a placental cell line that has been widely used as an in vitro model for the placenta. The b30 subclone of these cells can be grown on permeable membranes in bicameral chambers to form confluent cell layers, enabling rates of both nutrient uptake into the cells from the apical surface and efflux from the basolateral membrane to be determined. The aim of this study was to evaluate structural and functional properties of confluent b30 BeWo cell layers grown in bicameral chambers, focusing on the potential application for studying receptor-mediated uptake and transport of transferrin (Tf)-bound iron (Fe-Tf). While it proved extremely difficult to establish and maintain an intact BeWo cell monolayer, it was possible to grow the cells to a confluent multilayer. Iron, applied as Fe-Tf, was rapidly transported across this cell layer; 9.3 +/- 0.5% of the total dose was transported after 8 h, equivalent to 38.8 +/- 2.1 pmol.cm(-2).h(-1). Transfer of Tf across the cell layer was much more limited; 2.4 +/- 0.2% of the total dose was transported after 8 h, equivalent to 5.0 +/- 0.4 pmol.cm(-2).h(-1). Compartmental modeling of these data suggested that iron was transported across the cell layer predominantly, if not exclusively, via a transcellular route, whereas Tf taken up into the cells was predominantly recycled back to the apical compartment. The results suggest that these cells are very efficient at transporting iron and, under carefully controlled conditions, can be a valuable tool for the study of iron transport in the placenta.     

Apical and basolateral transferrin receptors in polarized BeWo cells recycle through separate endosomes

Contrary to most other epithelia, trophoblasts in the human placenta, which form the physical barrier between the fetal and the maternal blood circulation, express high numbers of transferrin receptors on their apical cell surface. This study describes the establishment of a polarized trophoblast-like cell line BeWo, which exhibit a high expression of transferrin receptors on the apex of the cells. Cultured on permeable filter supports, BeWo cells formed a polarized monolayer with microvilli on their apical cell surface. Across the monolayer a transepithelial resistance developed of approximately 600 omega.cm2 within 4 d. Depletion of Ca2+ from the medium decreased the resistance to background levels, showing its dependence on the integrity of tight junctions. Within the same period of time the secretion of proteins became polarized. In addition, the compositions of integral membrane proteins at the apical and basolateral plasma membrane domains were distinct as determined by domain-selective iodination. Similar to placental trophoblasts, binding of 125I-labeled transferrin to BeWo monolayers revealed that the transferrin receptor was expressed at both plasma membrane domains. Apical and basolateral transferrin receptors were found in a 1:2 surface ratio and exhibited identical dissociation constants and molecular weights. After uptake, transferrin recycled predominantly to the domain of administration, indicating separate recycling pathways from the apical and basolateral domain. This was confirmed by using diaminobenzidine cytochemistry, a technique by which colocalization of endocytosed 125I-labeled and HRP-conjugated transferrin can be monitored. No mixing of the two types of ligands was observed, when both ligands were simultaneously internalized for 10 or 60 min from opposite domains, demonstrating that BeWo cells possess separate populations of apical and basolateral early endosomes. In conclusion, the trophoblast-like BeWo cell line can serve as a unique model to compare the apical and basolateral endocytic pathways of a single ligand, transferrin, in polarized epithelial cells.     

Intracellular sorting and basolateral appearance of the G protein of vesicular stomatitis virus in Madin-Darby canine kidney cells

The polarity of the surface distribution of viral glycoproteins during virus infection has been studied in the Madin-Darby canine kidney epithelial cell line on nitrocellulose filters. Using a surface radioimmunoassay on Madin-Darby canine kidney strain I cells that had been infected with vesicular stomatitis virus or with avian influenza fowl plague virus, we found that the surface G protein was 97% basolateral, whereas the fowl plague virus hemagglutinin was 88% apical. Newly synthesized, pulse-labeled vesicular stomatitis virus appeared first on the basolateral plasma membrane as measured by an immunoprecipitation assay in which the anti-G protein antibody was applied to the monolayer either from the apical or the basolateral side. Labeled G protein could be accumulated inside the cell at a late stage of transport by decreasing the temperature to 20 degrees C during the chase. Reversal to 37 degrees C led to its rapid and synchronous transport to the basolateral surface at an initial rate 61-fold greater than that of transport to the apical side. These results demonstrate that the newly synthesized G protein is transported directly to the basolateral membrane and does not pass over the apical membrane en route. Since a previous study of the surface appearance of influenza virus hemagglutinins showed that the newly synthesized hemagglutinins were inserted directly from an intracellular site into the apical membrane (Matlin, K., and K. Simons, 1984, J. Cell Biol., 99:2131-2139), we conclude that the divergence of the transport pathway for the apical and basolateral viral glycoproteins has to occur intracellularly, i.e., before reaching the cell surface.     

A polarized human endometrial cell line that binds and transports polymeric IgA

Summary We have demonstrated that a human endometrial cell line, HEC-1, maintains a high transepithelial electrical resistance, directionally transports fluids across the cell monolayer, and releases enveloped viruses at distinct plasma membrane domains: influenza virus is released at the apical surfaces and vesicular stomatitis virus (VSV) at the basolateral surfaces. In addition, we have examined the expression of domain-specific endogenous proteins, including the polyimmunoglobulin receptor. Multiple endogenous polypeptides were found to be secreted into the culture medium at basolateral surfaces, whereas no secretion of specific polypeptides was observed from apical cell surfaces. Distinct patterns of endogenous proteins were also observed on apical and basolateral cell surfaces, with a much more complex polypeptide pattern on the basolateral membranes. Using surface biotinylation and immunofluorescence, the polyimmunoglobulin receptor was found to be expressed on the basolateral surfaces of HEC-1 monolayers. The specific binding of poly-immunoglobulin A (pIgA) was found to occur on the basolateral surface, and was followed by transcytosis to the apical surface and release into the apical medium. The observed characteristics indicate that the endometrium-derived HEC-1 epithelial cell line can be employed as a model for studies of protein transport in polarized epithelial cells of human endometrial tissues, as well as for studies of the interaction of microorganisms with epithelial cells in the genital tract.     

Distinct functional roles for the Menkes and Wilson copper translocating P-type ATPases in human placental cells.

BACKGROUND/AIMS: The copper transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND) are essential for normal copper transport in the human body. The placenta is the key organ in copper supply to the fetus during pregnancy and it is one of the few organs in the body to express both of the ATPases. The placenta therefore provides a unique opportunity to elucidate the specific roles of these transporters within the one cell type. METHODS/RESULTS: Using polarized placental Jeg-3 cells, siRNA technology and radio-labelled 64Cu transport assays, MNK and WND were shown to have distinct roles in the vectorial transport of copper. MNK transported copper from the cell via the basolateral membrane and in contrast, WND transported copper from the apical membrane. Inactivation of MNK resulted in decreased activity of two important cuproenzymes, lysyl oxidase and Cu/Zn-superoxide dismutase. CONCLUSIONS: Overall, these results provide definitive evidence for distinct roles of MNK and WND in the human placenta, and are consistent with a role for MNK in the transport of copper into the fetal circulation, and through delivery of copper to placental cuproenzymes, whilst WND contributes to the maintenance of placental copper homeostasis by transporting copper to the maternal circulation.     

Transport of micro-opioid receptor agonists and antagonist peptides across Caco-2 monolayer

Milk is the source of β-casomorphins – biologically active peptides with opioid activity – which are suspected to play various roles in the human body. The local influence of exogenous opioid peptides on gastrointestinal functions has been widely reported. After passing the gut barrier, β-casomorphins may affect the functions of immunological system, as well as dopaminergic, serotoninergic and GABA-ergic systems in brain, regulate the opioid receptor development and elicit behavioral effects. However, possibilities and mechanisms of the intestinal transport of β-casomorphins in human body in vivo have not been reported so far. In our research, the transepithelial transport of μ-opioid receptor agonists – human β-casomorphin-5 and 7(BCM5, BCM7) and antagonist – lactoferroxin A (LCF A) have been investigated using Caco-2 monolayer. In order to determine the pathway of investigated peptide transport across Caco-2 monolayer, two directions of the transport (apical to basolateral and basolateral to apical) have been studied. All investigated peptides were transported across the human intestinal cell line Caco-2 and the curves of cumulative amount of transported peptides in time were linear in each case. In addition, the hydrolysis of β-casomorphins during 60 min of experiment by dipeptidyl peptidase IV was observed. The data suggest the possibility of transport of opioid peptides derived from food across human intestinal mucosa.     

Phosphate transport by reconstructed monolayers from cultured mouse kidney cells

A reconstructed monolayer was formed using epithelial cells from normal mouse kidney to investigate the hormonal effect on phosphate transport by the renal cells. The cells, when cultured on a Millipore filter, formed a monolayer with an apical negative transepithelial potential of 8.4 +/- 0.4 mV. When radioactive phosphate was added onto the apical surface of the monolayer (corresponding to the luminal surface of a renal tubule), the phosphate was transported through the cell layer to the basolateral surface (corresponding to the peritubular surface of a renal tubule). This transport process was saturable, energy-dependent, and inhibited by 2,4-dinitrophenol or ouabain. Dose-dependent parathyroid hormone-induced inhibition (73% of the control) was also evident in this system. Similar inhibition (69% of the control) was observed with DBcAMP. Thus, monolayers reconstructed from cultured mouse kidney cells show characteristics similar to those of renal tubules.     

Hormonal regulation of the Menkes and Wilson copper-transporting ATPases in human placental Jeg-3 cells

Copper deficiency during pregnancy results in early embryonic death and foetal structural abnormalities including skeletal, pulmonary and cardiovascular defects. During pregnancy, copper is transported from the maternal circulation to the foetus by mechanisms which have not been clearly elucidated. Two copper-transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND), are expressed in the placenta and both are involved in placental copper transport, as copper accumulates in the placenta in both Menkes and Wilson disease. The regulatory mechanisms of MNK and WND and their exact role in the placenta are unknown. Using a differentiated polarized Jeg-3 cell culture model of placental trophoblasts, MNK and WND were shown to be expressed within these cells. Distinct roles for MNK and WND are suggested on the basis of their opposing responses to insulin. Insulin and oestrogen increased both MNK mRNA and protein levels, altered the localization of MNK towards the basolateral membrane in a copper-independent manner, and increased the transport of copper across this membrane. In contrast, levels of WND were decreased in response to insulin, and the protein was located in a tight perinuclear region, with a corresponding decrease in copper efflux across the apical membrane. These results are consistent with a model of copper transport in the placenta in which MNK delivers copper to the foetus and WND returns excess copper to the maternal circulation. Insulin and oestrogen stimulate copper transport to the foetus by increasing the expression of MNK and reducing the expression of WND. These data show for the first time that MNK and WND are differentially regulated by the hormones insulin and oestrogen in human placental cells.     

Regulation of Human Trophoblast GLUT1 Glucose Transporter by Insulin-Like Growth Factor I (IGF-I)

Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth.     

Intercellular transport of lysosomal acid lipase mediates lipoprotein cholesteryl ester metabolism in a human vascular endothelial cell-fibroblast coculture system.

We present results from studies of human cell culture models to support the premise that the extracellular transport of lysosomal acid lipase has a function in lipoprotein cholesteryl ester metabolism in vascular tissue. Vascular endothelial cells secreted a higher fraction of cellular acid lipase than did smooth muscle cells and fibroblasts. Acid lipase and lysosomal beta-hexosaminidase were secreted at approximately the same rate from the apical and basolateral surface of an endothelial cell monolayer. Stimulation of secretion with NH4Cl did not affect the polarity. We tested for the ability of secreted endothelial lipase to interact with connective tissue cells and influence lipoprotein cholesterol metabolism in a coculture system in which endothelial cells on a micropore filter were suspended above a monolayer of acid lipase-deficient (Wolman disease) fibroblasts. After 5-7 d, acid lipase activity in the fibroblasts reached 10%-20% of the level in normal cells; cholesteryl esters that had accumulated from growth in serum were cleared. Addition of mannose 6-phosphate to the coculture medium blocked acid lipase uptake and cholesterol clearance, indicating that lipase released from endothelial cells was packaged into fibroblast lysosomes by a phosphomannosyl receptor-mediated pathway. Supplementation of the coculture medium with serum was not required for lipase uptake and cholesteryl ester hydrolysis by the fibroblasts, but was necessary for cholesterol clearance. Results from our coculture model suggest that acid lipase may be transported from intact endothelium to cells in the lumen or the wall of a blood vessel. We postulate that delivery of acid hydrolases and lipoproteins to a common endocytic compartment may occur and have an impact on cellular lipoprotein processing.     

Effect of lectins on the transport of food ingredients in Caco-2 cell cultures

We investigated the effect of several lectins, such as soy bean lectin (SBA), concanavalin A (Con A), and wheat germ agglutinin (WGA), on the transport of some food ingredients (isoflavones, quercetin glycosides, carnosine/anserine) across Caco-2 cell monolayers. After incubation of food ingredients (0.03 approximately 2 mmol/L) in the presence or absence of lectins (1 approximately 180 microg/ml) on the apical side, aliquots were taken from the apical and basolateral solution, and were subjected to HPLC analysis. We also examined the effect of lectins on the permeability of the tight junction by measuring the transepithelial electrical resistance (TER) value of the Caco-2 cell monolayer. Isoflavones, which was not transported to the basolateral solution without lectins, could be transported in the presence of lectins, whereas their aglycones were detected at the same levels with or without the lectin treatment. The transport of quercetin glycosides also increased in the presence of lectins, however, that of peptides was not affected by the lectins. Con A and WGA, but SBA, decreased the TER value, indicating that Con A and WGA increased the transport via paracellular pathway, whereas SBA did via a different pathway.     

Sorting of newly synthesized galactosphingolipids to the two surface domains of epithelial cells

The high concentration of glycosphingolipids on the apical surface of epithelial cells may be generated by selective transport from their site of synthesis to the cell surface. Previously, we showed that canine kidney MDCK and human intestinal Caco-2 cells converted a ceramide carrying the short fluorescent fatty acid C6-NBD to glucosylceramide (GlcCer) and sphingomyelin (SM), and that GlcCer was preferentially transported to the apical surface as compared to SM. Here, we address the point that not all glycosphingolipid classes are apically enriched in epithelia. We show that a ceramide containing the 2-hydroxy fatty acid C6OH was preferentially converted by MDCK and Caco- 2 cells to galactosylceramide (GalCer) and its derivatives galabiosylceramide (Ga2Cer) and sulfatide (SGalCer) as compared to SM and GlcCer--all endogenous lipid classes of these cells. Transport to the apical and basolateral cell surface was monitored by a BSA- depletion assay. In MDCK cells, GalCer reached the cell surface with two- to sixfold lower apical/basolateral polarity than GlcCer. Remarkably, in Caco-2 cells GalCer and GlcCer displayed the same apical/basolateral polarity, but it was sixfold lower for lipids with a C6OH chain than for C6-NBD lipids. Therefore, the sorting of a sphingolipid appears to depend on lipid structure and cell type. We propose that the different ratios of gluco- and galactosphingolipid synthesis in the various epithelial tissues govern lipid sorting in the membrane of the trans Golgi network by dictating the composition of the domains from where vesicles bud to the apical and basolateral cell surface.     

Asymmetrical regulation of scavenger receptor class B type I by apical and basolateral stimuli using Caco-2 cells

Cholesterol uptake and the mechanisms that regulate cholesterol translocation from the intestinal lumen into enterocytes remain for the most part unclear. Since scavenger receptor class B type I (SR-BI) has been suggested to play a role in cholesterol absorption, we investigated cellular SR-BI modulation by various potential effectors administered in both apical and basolateral sides of Caco-2 cells. With differentiation, Caco-2 cells increased SR-BI protein expression. Western blot analysis showed the ability of cholesterol and oxysterols in both cell compartments to reduce SR-BI protein expression. Among the n-3, n-6, and n-9 fatty acid families, only eicosapentaenoic acid was able to lower SR-BI protein expression on both sides, whereas apical alpha-linolenic acid decreased SR-BI abundance and basolateral arachidonic acid (AA) raised it. Epidermal growth factor and growth hormone, either in the apical or basolateral medium, diminished SR-BI cellular content, while insulin displayed the same effect only on the basolateral side. In the presence of proinflammatory agents (LPS, TNF-alpha, IFN-gamma), Caco-2 cells exhibited differential behavior. SR-BI was downregulated by lipopolysaccharide on both sides. Finally, WY-14643 fibrate diminished SR-BI protein expression when it was added to the apical medium. Biotinylation studies in response to selected stimuli revealed that regulatory modifications in SR-BI protein expression occurred for the most part at the apical cell surface irrespective of the effector location. Our data indicate that various effectors supplied to the apical and basolateral compartments may impact on SR-BI at the apical membrane, thus suggesting potential regulation of intestinal cholesterol absorption and distribution in various intracellular pools.     

Fluid transport across cultured rat alveolar epithelial cells: a novel in vitro system

Previous studies have used fluid-instilled lungs to measure net alveolar fluid transport in intact animal and human lungs. However, intact lung studies have two limitations: the contribution of different distal lung epithelial cells cannot be studied separately, and the surface area for fluid absorption can only be approximated. Therefore, we developed a method to measure net vectorial fluid transport in cultured rat alveolar type II cells using an air-liquid interface. The cells were seeded on 0.4-microm microporous inserts in a Transwell system. At 96 h, the transmembrane electrical resistance reached a peak level (1,530 +/- 115 Omega.cm(2)) with morphological evidence of tight junctions. We measured net fluid transport by placing 150 microl of culture medium containing 0.5 microCi of (131)I-albumin on the apical side of the polarized cells. Protein permeability across the cell monolayer, as measured by labeled albumin, was 1.17 +/- 0.34% over 24 h. The change in concentration of (131)I-albumin in the apical fluid was used to determine the net fluid transported across the monolayer over 12 and 24 h. The net basal fluid transport was 0.84 microl.cm(-2).h(-1). cAMP stimulation with forskolin and IBMX increased fluid transport by 96%. Amiloride inhibited both the basal and stimulated fluid transport. Ouabain inhibited basal fluid transport by 93%. The cultured cells retained alveolar type II-like features based on morphologic studies, including ultrastructural imaging. In conclusion, this novel in vitro system can be used to measure net vectorial fluid transport across cultured, polarized alveolar epithelial cells.     

Permeation of hesperidin glycosides across Caco-2 cell monolayers via the paracellular pathway

The intestinal permeability to hesperidin glycosides was investigated by using a cultured monolayer of Caco-2 as a model for the small intestinal epithelium. Hesperidin glycosides were added to the apical side of the monolayer, and the substances that permeated to the basolateral side were determined by HPLC. Whereas hesperidin did not permeate across the Caco-2 monolayer, probably owing to its low solubility, the hesperidin glycosides did permeate. The transepithelial transport of hesperidin glycosides occurred in time- and dose-dependent manners. The transport was observed to be energy-independent, and was inversely correlated with the transepithelial electrical resistance (TEER) of the monolayer. These results suggest that hesperidin glycosides permeate across the Caco-2 cell monolayer via the paracellular pathway.     

Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells

We characterized the three-dimensional organization of microtubules in the human intestinal epithelial cell line Caco-2 by laser scanning confocal microscopy. Microtubules formed a dense network approximately 4-microns thick parallel to the cell surface in the apical pole and a loose network 1-micron thick in the basal pole. Between the apical and the basal bundles, microtubules run parallel to the major cell axis, concentrated in the vicinity of the lateral membrane. Colchicine treatment for 4 h depolymerized 99.4% of microtubular tubulin. Metabolic pulse chase, in combination with domain-selective biotinylation, immune and streptavidin precipitation was used to study the role of microtubules in the sorting and targeting of four apical and one basolateral markers. Apical proteins have been recently shown to use both direct and transcytotic (via the basolateral membrane) routes to the apical surface of Caco-2 cells. Colchicine treatment slowed down the transport to the cell surface of apical and basolateral proteins, but the effect on the apical proteins was much more drastic and affected both direct and indirect pathways. The final effect of microtubular disruption on the distribution of apical proteins depended on the degree of steady-state polarization of the individual markers in control cells. Aminopeptidase N (APN) and sucrase-isomaltase (SI), which normally reach a highly polarized distribution (110 and 75 times higher on the apical than on the basolateral side) were still relatively polarized (9 times) after colchicine treatment. The decrease in the polarity of APN and SI was mostly due to an increase in the residual basolateral expression (10% of control total surface expression) since 80% of the newly synthesized APN was still transported, although at a slower rate, to the apical surface in the absence of microtubules. Alkaline phosphatase and dipeptidylpeptidase IV, which normally reach only low levels of apical polarity (four times and six times after 20 h chase, nine times and eight times at steady state) did not polarize at all in the presence of colchicine due to slower delivery to the apical surface and increased residence time in the basolateral surface. Colchicine-treated cells displayed an ectopic localization of microvilli or other apical markers in the basolateral surface and large intracellular vacuoles. Polarized secretion into apical and basolateral media was also affected by microtubular disruption. Thus, an intact microtubular network facilitates apical protein transport to the cell surface of Caco-2 cells via direct and indirect routes; this role appears to be crucial for the final polarity of some apical plasma membrane proteins but only an enhancement factor for others.     

Effect of endothelial cell polarity on beta-amyloid-induced migration of monocytes across normal and AD endothelium

During normal aging and amyloid beta-peptide (Abeta) disorders such as Alzheimer's disease (AD), one finds increased deposition of Abeta and activated monocytes/microglial cells in the brain. Our previous studies show that Abeta interaction with a monolayer of normal human brain microvascular endothelial cells results in increased adherence and transmigration of monocytes. Relatively little is known of the role of Abeta accumulated in the AD brain in mediating trafficking of peripheral blood monocytes (PBM) across the blood-brain barrier (BBB) and concomitant accumulation of monocytes/microglia in the AD brain. In this study, we showed that interaction of Abeta(1--40) with apical surface of monolayer of brain endothelial cells (BEC), derived either from normal or AD individuals, resulted in increased transendothelial migration of monocytic cells (HL-60 and THP-1) and PBM. However, transmigration of monocytes across the BEC monolayer cultivated in a Transwell chamber was increased 2.5-fold when Abeta was added to the basolateral side of AD compared with normal individual BEC. The Abeta-induced transmigration of monocytes was inhibited in both normal and AD-BEC by antibodies to the putative Abeta receptor, receptor for advanced glycation end products (RAGE), and to the endothelial cell junction molecule, platelet-endothelial cell adhesion molecule-1 (PECAM-1). We conclude that interaction of Abeta with the basolateral surface of AD-BEC induces cellular signaling, promoting transmigration of monocytes from the apical to basolateral direction. We suggest that Abeta in the AD brain parenchyma or cerebrovasculature initiates cellular signaling that induces PBM to transmigrate across the BBB and accumulate in the brain.     

Microtubule perturbation retards both the direct and the indirect apical pathway but does not affect sorting of plasma membrane proteins in intestinal epithelial cells (Caco-2).

Endogenous plasma membrane proteins are sorted from two sites in the human intestinal epithelial cell line Caco-2. Apical proteins are transported from the Golgi apparatus to the apical domain along a direct pathway and an indirect pathway via the basolateral membrane. In contrast, basolateral proteins never appear in the apical plasma membrane. Here we report on the effect of the microtubule-active drug nocodazole on the post-synthetic transport and sorting of plasma membrane proteins. Pulse-chase radiolabeling was combined with domain-specific cell surface assays to monitor the appearance of three apical and one basolateral protein in plasma membrane domains. Nocodazole was found to drastically retard both the direct transport of apical proteins from the Golgi apparatus and the indirect transport (transcytosis) from the basolateral membrane to the apical cell surface. In contrast, neither the transport rates of the basolateral membrane nor the sorting itself were significantly affected by the nocodazole treatment. We conclude that an intact microtubular network facilitates, but is not necessarily required for, the transport of apical membrane proteins along the two post-Golgi pathways to the brush border.     

Decreased hephaestin expression and activity leads to decreased iron efflux from differentiated Caco2 cells

Iron is transported across intestinal brush border cells into the circulation in at least two distinct steps. Iron can enter the enterocyte via the apical surface through several paths. However, iron egress from the basolateral side of enterocytes converges on a single export pathway requiring the iron transporter, ferroportin1, and hephaestin, a ferroxidase. Copper deficiency leads to reduced hephaestin protein expression and activity in mouse enterocytes and intestinal cell lines. We tested the effect of copper deficiency on differentiated Caco2 cells grown in transwells and found decreased hephaestin protein expression and activity as well as reduced ferroportin1 protein levels. Furthermore, the decrease in hephaestin levels correlates with a decrease of ⁵⁵Fe release from the basolateral side of Caco2 cells. Presence of ceruloplasmin, apo‐transferrin or holo‐transferrin did not significantly alter the results observed. Repletion of copper in Caco2 cells leads to reconstitution of hephaestin protein expression, activity, and transepithelial iron transport. J. Cell. Biochem. 107: 803–808, 2009. © 2009 Wiley‐Liss, Inc.