Glucose and calcium ions may modulate the efficiency of bovine β-casomorphin-7 permeability through a monolayer of Caco-2 cells

Milk and dairy products provide a lot of valuable nutritive elements. They are also sources of biologically active peptides, including β-casomorphins that manifest the properties of morphine. An activity of DPPIV seems to be most crucial factor decreasing the efficiency of the β-casomorphin-7 (BCM7) transport. The increase of BCM7 concentration in blood may intensify symptoms of apparent life threatening events (ALTE), autism, schizophrenia, and allergy. This study aimed at identifying the influence of several selected substances on a transport efficiency of bovine BCM7 through an intestinal monolayer in a Caco-2 cell model system. Applying the ELISA method, the permeability coefficient of BCM7 through the Caco-2 monolayer was calculated. TEER values were used to evaluate the integrity of Caco-2 cell monolayers. An increase of glucose and Ca²⁺ concentrations in the culture medium was accompanied by an increase of the BCM7 transport efficiency. The lowest permeability coefficients of BCM7 were observed for the membranes with high electrical resistances. The transport was enhanced in the presence of milk infant formulas, whereas no changes were observed when using μ-opioid receptor antagonist (casoxin-6). The results may be useful in understanding the pathogenesis of inflammation and food allergy in infants.     

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.     

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.     

Differential effects of basolateral and apical iron supply on iron transport in Caco-2 cells

Iron homeostasis in the human body is maintained primarily through regulation of iron absorption in the duodenum. The liver peptide hepcidin plays a central role in this regulation. Additionally, expression and functional control of certain components of the cellular iron transport machinery can be influenced directly by the iron status of enterocytes. The significance of this modulation, relative to the effects of hepcidin, and the comparative effects of iron obtained directly from the diet and/or via the bloodstream are not clear. The studies described here were performed using Caco-2 cell monolayers as a model of intestinal epithelium, to compare the effects of iron supplied in physiologically relevant forms to either the apical or basolateral surfaces of the cells. Both sources of iron provoked increased cellular ferritin content, indicating iron uptake from both sides of the cells. Supply of basolateral transferrin-bound iron did not affect subsequent iron transport across the apical surface, but reduced iron transport across the basolateral membrane. In contrast, the apical iron supply led to subsequent reduction in iron transport across the apical cell membrane without altering iron export across the basolateral membrane. The apical and basolateral iron supplies also elicited distinct effects on the expression and subcellular distribution of iron transporters. These data suggest that, in addition to the effects of cellular iron status on the expression of iron transporter genes, different modes and direction of iron supply to enterocytes can elicit distinct functional effects on iron transport.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0463-5) contains supplementary material, which is available to authorized users.     

Insulin and IGF-1 Receptors in A Human Intestinal Adenocarcinoma Cell Line (Caco-2): Regulation of Na+ Glucose Transport Across the Brush Border

Abstract

Both insulin and IGF–1 receptors are present in intestinal mucosal cells, although their role in this tissue is unclear. We have characterized these receptors in a human adenocarcinoma cell line, Caco-2, and examined their role in the regulation of glucose transport and absorption in these cells. The Caco-2 cells demonstrated specific insulin and IGF-1 receptors. They also bound cytochalasin B, suggesting the presence of a glucose transporter-like protein. When grown on membranes, the Caco-2 cells formed columnar, bipolar cells with tight junctions. The monolayer selectively transported D-glucose, and methyl-D-glucose, with complete exclusion of L-glucose, D-mannitol and inulin. The absorption of glucose across the monolayer occurred via a Na⁺/glucose cotransporter, as indicated by a change in short circuit current after addition of glucose to the apical membrane. When examined under several conditions, neither insulin nor IGF-1 had an affect on the transport of glucose across the Caco-2 monolayer, nor the production of lactate by the cells. It is concluded that the insulin and IGF-1 receptors of Caco-2 cells do not regulate glucose transport.     

Transport of cholesterol across a BeWo cell monolayer: implications for net transport of sterol from maternal to fetal circulation

The placental transport of various compounds, such as glucose and fatty acids, has been well studied. However, the transport of cholesterol, a sterol essential for proper fetal development, remains undefined in the placenta. Therefore, the purpose of these studies was to examine the transport of cholesterol across a placental monolayer and its uptake by various cholesterol acceptors. BeWo cells, which originated from a human choriocarcinoma, were grown on transwells for 3 days to form a confluent monolayer. The apical side of the cells was radiolabeled with either free cholesterol or LDL cholesteryl ester. After 24 h, the radiolabel was removed and cholesterol acceptors were added to the basolateral chamber. Cholesterol was found to be taken up by the apical surface of the placental monolayer, transported to the basolateral surface of the cell, and effluxed to fetal human serum, fetal HDL, or phospholipid vesicles, but not to apolipoprotein A-I. In addition, increasing the cellular cholesterol concentration further increased the amount of cholesterol transported to the basolateral acceptors. These are the first studies to demonstrate the movement of cholesterol across a placental cell from the maternal circulation (apical side) to the fetal circulation (basolateral side).     

Transport of sennosides and sennidines from Cassia angustifolia and Cassia senna across Caco-2 monolayers – an in vitro model for intestinal absorption

Laxative effects of Senna preparations are mainly mediated by rheinanthrone, a metabolite formed in the intestinal flora from dianthrones. Nevertheless, it was not clear whether dianthrones are bioavailable at all and contribute to the overall effects of this important medicinal plant. Using the Caco-2 human colonic cell line as an in vitro model of the human intestinal mucosal barrier, the bioavailability of dianthrones was studied in apical to basolateral (absorptive) and basolateral to apical (secretive) direction. Permeability coefficients (P_c) and percent transport were calculated based on quantitations by HPLC. From the data obtained it was concluded that sennosides A and B, as well as their aglycones sennidine A and B are transported through the Caco-2 monolayers in a concentration-dependent manner and their transport was linear with time. The absorption in apical to basolateral direction was poor and P_c values were comparable to mannitol. The transport was higher in the secretory direction, indicating a significant efflux (e.g. by efflux pumps) of the (poorly) absorbed compounds in the intestinal lumen again. Our findings support the general understanding that the laxative effects of Senna are explainable mainly by metabolites and not by the natively present dianthrones.     

Transepithelial transport of the bioactive tripeptide, Val-Pro-Pro, in human intestinal Caco-2 cell monolayers

Some of the food-derived tripeptides with angiotensin converting enzyme (ACE)-inhibitory activity have been reported to be hypotensive after being orally administered. The mechanism for the intestinal transport of these tripeptides was studied by using monolayer-cultured human intestinal Caco-2 cells which express many enterocyte-like functions including the peptide transporter (PepT1)-mediated transport system. Val-Pro-Pro, an ACE-inhibitory peptide from fermented milk, was used as a model tripeptide. A significant amount of intact Val-Pro-Pro was transported across the Caco-2 cell monolayer. This transport was hardly inhibited by a competitive substrate for PepT1. Since no intact Val-Pro-Pro was detected in the cells, Val-Pro-Pro apically taken by Caco-2 cells via PepT1 was likely to have been quickly hydrolyzed by intracellular peptidases, producing free Val and Pro. These findings suggest that PepT1-mediated transport was not involved in the transepithelial transport of intact Val-Pro-Pro. Paracellular diffusion is suggested to have been the main mechanism for the transport of intact Val-Pro-Pro across the Caco-2 cell monolayer.     

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.     

Extracellular metabolism-dependent uptake of lysolipids through cultured monolayer of differentiated Caco-2 cells

Glycerophospholipids are known to be hydrolyzed in the intestinal lumen into free fatty acids and lysophospholipids that are then absorbed by the intestinal epithelial cells. A monolayer of enterocyte-differentiated Caco-2 cell is often used to assess the intestinal bioavailability of nutrients. In this study, we examined how differentiated Caco-2 cells process lysoglycerolipids such as lysophosphatidylcholine (LPC). Our findings were twofold. (1) Caco-2 cells secreted both a lysophospholipase A-like enzyme and a glycerophosphocholine-phosphodiesterase enzyme into the apical, but not basolateral, lumen, suggesting that food-derived LPC is converted to a free fatty acid, sn-glycerol-3-phosphate, and choline through two sequential enzymatic reactions in humans. The release of the latter enzyme was differentiation-dependent. (2) Fatty acid-releasing activities toward exogenous fluorescent LPC, lysophosphatidic acid and monoacylglycerol were shown to be higher on the apical membranes of Caco-2 cells than on the basolateral membranes. These results suggest that human intestinal epithelial cells metabolize lysoglycerolipids by two distinct mechanisms involving secreted or apical-selective expression of metabolic enzymes.     

Chemical Form of Selenium Affects Its Uptake,Transport, and Glutathione Peroxidase Activity in the Human Intestinal Caco-2 Cell Model

Determining the effect of selenium (Se) chemical form on uptake, transport, and glutathione peroxidase activity in human intestinal cells is critical to assess Se bioavailability at nutritional doses. In this study, we found that two sources of L-selenomethionine (SeMet) and Se-enriched yeast each increased intracellular Se content more effectively than selenite or methylselenocysteine (SeMSC) in the human intestinal Caco-2 cell model. Interestingly, SeMSC, SeMet, and digested Se-enriched yeast were transported at comparable efficacy from the apical to basolateral sides, each being about 3-fold that of selenite. In addition, these forms of Se, whether before or after traversing from apical side to basolateral side, did not change the potential to support glutathione peroxidase (GPx) activity. Although selenoprotein P has been postulated to be a key Se transport protein, its intracellular expression did not differ when selenite, SeMSC, SeMet, or digested Se-enriched yeast was added to serum-contained media. Taken together, our data show, for the first time, that the chemical form of Se at nutritional doses can affect the absorptive (apical to basolateral side) efficacy and retention of Se by intestinal cells; but that, these effects are not directly correlated to the potential to support GPx activity.     

Copper repletion enhances apical iron uptake and transepithelial iron transport by Caco-2 cells

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7-8 days in media supplemented with 1 microM CuCl(2) had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.     

Transport and function of syntaxin 3 in human epithelial intestinal cells

To follow the transport of human syntaxin (Syn) 3 to theapical surface of intestinal cells, we produced and expressed in Caco-2cells a chimera made of the entire Syn3 coding sequence and theextracellular domain of the human transferrin receptor (TfR). Thischimera (Syn3TfR) was localized to the apical membrane and wastransported along the direct apical pathway, suggesting that this isalso the case for endogenous Syn3. To test the potential role of Syn3in apical transport, we overexpressed it in Caco-2 cells and measuredthe efficiency of apical and basolateral delivery of several endogenousmarkers. We observed a strong inhibition of apical delivery ofsucrase-isomaltase (SI), an apical transmembrane protein, and of-glucosidase, an apically secreted protein. No effect was observedon the basolateral delivery of Ag525, a basolateral antigen, stronglysuggesting that Syn3 is necessary for efficient delivery of proteins tothe apical surface of intestinal cells.

     

Heme transport exhibits polarity in Caco-2 cells: evidence for an active and membrane protein-mediated process

Heme prosthetic groups are vital for all living organisms, but they can also promote cellular injury by generating reactive oxygen species. Therefore, intestinal heme absorption and distribution should be carefully regulated. Although a human intestine brush-border heme receptor/transporter has been suggested, the mechanism by which heme crosses the apical membrane is unknown. After it enters the cell, heme is degraded by heme oxygenase-1 (HO-1), and iron is released. We hypothesized that heme transport is actively regulated in Caco-2 cells. Cells exposed to hemin from the basolateral side demonstrated a higher HO-1 induction than cells exposed to hemin from the apical surface. Hemin secretion was more rapid than absorption, and net secretion occurred against a concentration gradient. Treatment of the apical membrane with trypsin increased hemin absorption by threefold, but basolateral treatment with trypsin had no effect on hemin secretion. Neither apical nor basolateral trypsin changed the paracellular pathway. We conclude that heme is acquired and transported in both absorptive and secretory directions in polarized Caco-2 cells. Secretion is via an active metabolic/transport process. Trypsin applied to the apical surface increased hemin absorption, suggesting that protease activity can uncover a process for heme uptake that is otherwise quiescent. These processes may be involved in preventing iron overload in humans.     

Identification of signals and mechanisms of sorting of plasma membrane proteins in intestinal epithelial cells]

In epithelial cells the plasma membrane is divided into domains that are biochemically and functionally different. In intestinal cells for example the apical domain is facing the intestinal lumen and is involved in the uptake of nutriments while the basolateral domain is mediating cell-cell adhesion and signalisation. We are interested in deciphering the mechanisms underlying the creation and maintenance of such specialized domains. As an epithelial model we have used the intestinal cell line Caco-2 and we have studied the transport and sorting of the human neurotrophin receptor (p75 NTR) in these cells. Newly synthesized p75 NTR is first transported to the basolateral membrane and then is accumulated on the apical membrane after transcytosis. This final apical localization is controlled by the presence of a membrane anchor and a cluster of O-glycosylation sites located in the part of the ectodomain close to the membrane. Among the mechanisms likely to be involved in the sorting of apical components we have looked for a role of lipid-protein microdomain formation in the Golgi apparatus. These membrane microdomains are highly enriched in glycosylphosphatidyl inositol (GPI) anchored proteins, glycosphingolipids and apical proteins such as sucrase isomaltase (SI). Such a composition is also found for endocytic structures called caveolae which are made of caveolin 1. We have expressed caveolin 1 in Caco-2 cells which do not express it and also caveolin 2, a related protein of unknown function. Expression of caveolin 1 led to formation of caveolae indicating that this protein is necessary for caveolae formation while caveolin 2 is restricted to the Golgi apparatus and has no effect on caveolae formation. However Caveolin 2 increased the amount of SI incorporated in microdomains suggesting a role in recruitment into the apical pathway. The choice for a site of fusion for transport vesicles is the last step of control during exocytosis. To identify proteins involved in that step we have cloned and characterized two members of the t-SNARE family, namely syntaxin 3 and SNAP23. Syntaxin 3 is present on the apical membrane and forms a complex with SNAP23 which is also localized on the basolateral membrane where it forms a complex with syntaxin 4. Overexpression of syntaxin 3 in Caco-2 led to a decrease of SI exocytosis towards the apical membrane confirming that syntaxin 3 is involved in targeting the fusion of apical transport vesicles to the apical pole of the cells.     

Transepithelial Taurine Transport in Caco-2 Cell Monolayers

Here we characterized transepithelial taurine transport in monolayers of cultured human intestinal Caco-2 cells by analyzing kinetic apical and basolateral uptake and efflux parameters. Basolateral uptake was Na⁺- and Cl⁻- dependent and was inhibited by β-amino acids. Uptake by this membrane showed properties similar to those of the apical TauT system. In both membranes, taurine uptake fitted a model consisting of a non-saturable plus a saturable component, with a higher half-saturation constant and transport capacity at the apical membrane (K_m, 17.1 μmol/L; V_max, 28.4 pmol·cm⁻²·5 min⁻¹) than in the basolateral domain (K_m, 9.46 μmol/L; V_max, 5.59 pmol·cm⁻²·5 min⁻¹). The non-saturable influx component, estimated in the absence of Na⁺ and Cl⁻, showed no significant differences between apical and basolateral membranes (K_D, 89.2 and 114.7 nL·cm⁻² · 5 min⁻¹, respectively). Taurine efflux from the cells is a diffusive process, as shown in experiments using preloaded cells and in trans-stimulation studies (apical K_D,72.7 and basolateral K_D, 50.1 nL·cm⁻²·5 min⁻¹). Basolateral efflux rates were significantly lower than passive influx rates. We conclude that basolateral taurine uptake in Caco-2 cells is mediated by a transport mechanism that shares some properties with the apical system TauT. Moreover, calculation of unidirectional and transepithelial taurine fluxes reveals that apical influx of this amino acid is higher than basolateral efflux rates, thereby enabling epithelial cells to accumulate taurine against a concentration gradient.     

Polarized expression of type I phosphodiesterases in epithelial cells]

Type I phosphodiesterases are differently expressed by different cell types. Three members have been identified, PC-1, B10 and autotaxin. They are between 40 and 50% identical at the amino acid sequence level. Hepatocytes express both B10 and PC-1 at their plasma membrane. However, B10 is exclusively expressed at the apical pole whereas PC-1 is located at the basolateral pole. Studies of the biosynthetic route of B10 in hepatocytes shows that B10 is first transported to the basolateral surface and secondarily reaches the basolateral surface. The transcytotic step between the basolateral and apical surface occurs through a tubular endosomal compartment identical to the transcytotic compartment of the polymeric IgA receptor. Transfected in the polarized cell lines MDCK and Caco-2 of renal and intestinal origin, B10 and PC-1 are expressed at the apical and basolateral poles respectively, as in hepatocytes. However, the biosynthetic transport of B10 occurs directly in MDCK cells and both directly and by transcytosis in Caco-2 cells. Truncation of the cytoplasmic domain of PC-1 generates an apical protein indicating that the basolateral signal of PC-1 is likely to be in the cytoplasmic domain. The nature of the apical targeting signal of B10 is under investigation.     

Docosahexaenoic acid and eicosapentaenoic acid-enriched phosphatidylcholine liposomes enhance the permeability, transportation and uptake of phospholipids in Caco-2 cells

The influence of docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-enriched phosphatidylcholine (PC) on the permeability, transport and uptake of phospholipids was evaluated in Caco-2 cells. The cells were grown on permeable polycarbonate transwell filters, thus allowing separate access to the apical and basolateral chambers. The monolayers of the cells were used to measure lucifer yellow permeability and transepithelial electrical resistance (TEER). Transcellular transportation of diphenylhexatriene (DPH) labeled-PC small unilamellar vesicles (SUV) from the apical to basolateral chamber, and uptake of the same SUV was monitored in the cell monolayers. Cell-membrane perturbation was evaluated to measure the release of lactate dehydrogenase and to determine the cell viability with sodium 2-(4-iodophenyl)-3-(4-nitrophenyl) -5-(2, 4-disulfophenyl)-2H-tetrazolium dye reduction assay. The lucifer yellow flux was 1.0 and 1.5 nmol/h/cm² with 50 μM PC, and 17.0 and 23.0 nmol/h/cm² with 100 μM PC when monolayers of Caco-2 cells were treated with DHA- and EPA-enriched PC, respectively. TEER decreased to 24 and 27% with 50 and 100 μM DHA-enriched PC, and to 25 and 30% with 50 and 100 μM EPA-enriched PC, respectively. Our results show that DHA- and EPA-enriched PC increases tight junction permeability across the Caco-2 cell monolayer whereas soy PC has no effect on tight junction permeability. Transportation and uptake of DHA- and EPA-enriched PC SUV differed significantly (P < 0.01) from those of soy PC SUV at all doses. We found that PC SUV transported across Caco-2 monolayer and was taken up by Caco-2 cells with very slight injury of the cell membrane up to 100 μM PC. Lactate dehydrogenase release and cell viability did not differ significantly between the treatment and control, emphasizing that injury was minimal. Our results suggest that DHA- and EPA-enriched PC enhance the permeability, transport and uptake of PC SUV across monolayers of Caco-2 cells. (Mol Cell Biochem xxx: 1–9, 2005)