Capsaicin-enhanced Ribosomal Protein P2 Expression in Human Intestinal Caco-2 Cells

On the basis of transepithelial electrical resistance (TER) measurements, we found that capsaicin (100 μM)-treated human intestinal Caco-2 cells show a momentary increase in tight-junction (TJ) permeability (decrease in TER) followed by a complete recovery. We used proteome analysis to search for proteins that are associated with the recovery of TJ permeability in capsaicin-treated Caco-2 cells. A protein with a relative molecular mass of 14 kDa was found to be expressed more highly in capsaicin-treated cells than in nontreated cells. Mass spectrometry and sequence analyses revealed that the protein that is expressed significantly upon capsaicin treatment is the ribosomal protein P2; its cDNA sequence was identical to that found in the human genome database. An increase in the amount of cellular filamentous actin (F-actin) was shown after 8 h of incubation with capsaicin. It has been reported that P2 activates elongation factor 2, which stabilizes F-actin filaments, and that the depolymerization of F-actin is associated with the increase in TJ permeability (decrease in TER). Consequently, these results suggest that P2 plays an important role in the recovery of the TJ permeability in capsaicin-treated human intestinal cells. An erratum to this article is available at .     

Na+-independent Lysine Transport in Human Intestinal Caco-2 Cells

The nature of transepithelial and cellular transport of the dibasic amino acid lysine in human intestinal epithelial Caco-2 cells has been characterized. Intracellular accumulation of lysine across both the apical and basolateral membranes consists of a Na⁺-independent, membrane potential-sensitive uptake. Na⁺-independent lysine uptake at the basolateral membrane exceeds that at the apical membrane. Lysine uptake consists of both saturable and nonsaturable components. Na⁺-independent lysine uptake at both membranes is inhibited by lysine, arginine, alanine, histidine, methionine, leucine, cystine, cysteine and homoserine. In contrast, proline and taurine are without inhibitory effects at both membranes. Fractional Na⁺-independent lysine efflux from preloaded epithelial layers is greater at the basolateral membrane and shows trans-stimulation across both epithelial borders by lysine, arginine, alanine, histidine, methionine, and leucine but not proline and taurine. Na⁺-independent lysine influx (10 μm) in the presence of 10 mm homoserine shows further concentration dependent inhibition by lysine. Taken together, these data are consistent with lysine transport being mediated by systems b^o,+, y⁺ and a component of very low affinity (nonsaturable) at both membranes. The relative contribution to lysine uptake at each membrane surface (at 10 μm lysine), normalized to total apical uptake (100%), is apical b^o,+ (47%), y⁺ (27%) and the nonsaturable component (26%), and basal b^o,+ (446%), y⁺ (276%) and the nonsaturable component (20%). Northern analysis shows hybridization of Caco-2 poly(A)⁺RNA with a human rBAT cDNA probe. Received: 3 July 1995/Revised: 6 February 1996     

Transepithelial Transport of Rosmarinic Acid in Intestinal Caco-2 Cell Monolayers

The absorption characteristics of rosmarinic acid (RA) were examined by measuring permeation across Caco-2 cell monolayers using an HPLC-electrochemical detector (ECD) fitted with a coulometric detection system. RA exhibited nonsaturable transport even at 30 mM, and the permeation at 5 mM in the apical-to-basolateral direction, J _ap→bl, was 0.13 nmol/min/mg of protein. This permeation rate is nearly the same as that of 5 mM chlorogenic acid (CLA) and gallic acid, which are paracellularly transported compounds. Almost all of the apically loaded RA was retained on the apical side, and J _ap→bl was inversely correlated with paracellular permeability. These results indicate that RA transport was mainly via paracelluar diffusion, and the intestinal absorption efficiency of RA was low. Furthermore, RA appeared to be unsusceptible to hydrolysis by mucosa esterase in Caco-2 cells. These results, together with our previous work (J. Agric. Food Chem., 52, 2518–2526 (2004), J. Agric. Food Chem., 52, 6418–6424 (2004)) suggest that the majority of RA is further metabolized and degraded into m-coumaric and hydroxylated phenylpropionic acids by gut microflora, which are then efficiently absorbed and distributed by the monocarboxylic acid transporter (MCT) within the body. The potential of orally administered RA in vivo will be further investigated.     

Lactobacillus casei NY1301 Increases the Adhesion of Lactobacillus gasseri NY0509 to Human Intestinal Caco-2 Cells

In the presence of Lactobacillus casei NY1301, the adhesion of Lactobacillus gasseri NY0509 to cultured human intestinal Caco-2 cells was significantly increased (P<0.01). In contrast, L. gasseri NY0509 did not affect the adhesion of L. casei NY1301. A heat-stable cell component of L. casei NY1301 was involved in this increase of adhesion. These results suggest that a combination of these strains may have synergistic effects of adhesion to human intestinal mucosa.     

A Mucus Adhesion Promoting Protein,MapA, Mediates the Adhesion of Lactobacillus reuteri to Caco-2 Human Intestinal Epithelial Cells

Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.     

Heme Iron Uptake by Caco-2 Cells is a Saturable,Temperature Sensitive and Modulated by Extracellular pH and Potassium

It is known that heme iron and inorganic iron are absorbed differently. Heme iron is found in the diet mainly in the form of hemoglobin and myoglobin. The mechanism of iron absorption remains uncertain. This study focused on the heme iron uptake by Caco-2 cells from a hemoglobin digest and its response to different iron concentrations. We studied the intracellular Fe concentration and the effect of time, K⁺ depletion, and cytosol acidification on apical uptake and transepithelial transport in cells incubated with different heme Fe concentrations. Cells incubated with hemoglobin-digest showed a lower intracellular Fe concentration than cells grown with inorganic Fe. However, uptake and transepithelial transport of Fe was higher in cells incubated with heme Fe. Heme Fe uptake had a low V _max and K _m as compared to inorganic Fe uptake and did not compete with non-heme Fe uptake. Heme Fe uptake was inhibited in cells exposed to K⁺ depletion or cytosol acidification. Heme oxygenase 1 expression increased and DMT1 expression decreased with higher heme Fe concentrations in the media. The uptake of heme iron is a saturable and temperature-dependent process and, therefore, could occur through a mechanism involving both a receptor and the endocytic pathway.     

Effect of long-term fluoxetine treatment on the human serotonin transporter in Caco-2 cells

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) broadly used in the treatment of human mood disorders and gastrointestinal diseases involving the serotoninergic system. The effectiveness of this therapy depends on repeated long-term treatment. Most of the long-term studies in vivo of SSRI effects on serotoninergic activity have focused on their effects on autoreceptors or postsynaptic receptors. The chronic effect of SSRIs on the activity of the serotonin transporter (SERT) has been less studied and the results have been contradictory. The aim of this study was to determine the specific effect of long-term fluoxetine treatment on human serotonin transporter (hSERT) in vitro, by using the human enterocyte-like cell line Caco-2. Results show that fluoxetine diminished the 5-HT uptake in a concentration-dependent way and that this effect was reversible. Fluoxetine affected mainly the hSERT transport rate by reducing the availability of the transporter in the membrane with no significant alteration of either the total hSERT protein content or the hSERT mRNA level. These results suggest that the effect of fluoxetine on the expression of hSERT is post-translational and has shown itself to be independent of PKC and PKA activity. This study may be useful to clarify the effect of the long-term fluoxetine therapy in both gastrointestinal and central nervous system disorders.     

Effects of Food Lectins on the Transport System of Human Intestinal Caco-2 Cell Monolayers

The effects of 16 lectins isolated from foodstuff on the transport system across human intestinal Caco-2 cell monolayers were investigated by using four fluorescent markers: lucifer yellow (LY) for the paracellular pathway, fluorescein (FL) for the monocarboxylic acid transporter-mediated pathway, rhodamine 123 for the P-glycoprotein-mediated efflux pathway, and calcein for the multidrug resistance associated protein-related efflux pathway. The transepithelial electrical resistance (TER) values for the monolayers were also measured. WGA from wheat germ, ABA from white mushroom, AOL from Aspergillus oryzae, and CSL3 from chum salmon eggs (each at 100 µg/mL) decreased the TER value by 20–40% which resulted in increased LY transport. These lectins, as well as such other lectins as SBA from soybean, RBA from rice bran, and Con A from jack bean, affected other transport pathways too. These results indicate that the lectins modulated the transepithelial transport system in different ways, probably because of their specific binding characteristics toward Caco-2 cell monolayers.     

大肠杆菌K88体外黏附Caco-2细胞及其对细胞膜的影响

采用体外Caco-2细胞培养模型,研究大肠杆菌K88黏附Caco-2肠上皮细胞后对其存活率及增殖活力、细胞膜磷脂酶A2、细胞内Ca^2 浓度及膜流动性的影响。结果表明,细菌黏附3h后细胞活力明显下降,PLA2活性升高,细胞内Ca^2 浓度增加,细胞膜流动性降低,从而导致肠上皮细胞膜结构和功能的损害。     

蒙脱石对细菌黏附Caco-2细胞的影响

采用Caco-2细胞培养模型,观察两歧双歧杆菌、嗜酸乳杆菌、嗜水气单胞菌、副溶血弧菌、大肠杆菌、鼠伤寒沙门菌的黏附率,并在培养液中加入蒙脱石,计算蒙脱石对细菌黏附的阻断率,探讨蒙脱石对上述细菌黏附作用的影响。结果表明:所试菌与Caco-2细胞均有不同程度的黏附作用;蒙脱石对细菌黏附Caco-2细胞均有不同程度的阻断作用,对病原菌黏附Caco-2细胞的阻断作用要明显大于其对益生菌的阻断效果,其中对大肠杆菌、鼠伤寒沙门菌、嗜水气单胞菌、副溶血弧菌黏附的阻断率分别为54.22%、48.41%、60.53%、50.64%,而对两歧双歧杆菌、嗜酸乳杆菌黏附的阻断率分别为25.64%和21.49%。结果提示蒙脱石可有效阻断病原菌黏附,从而防治肠道细菌感染和细菌移位。     

Cytokine Responses of Intestinal Epithelial-Like Caco-2 Cells to Non-Pathogenic and Opportunistic Pathogenic Yeasts in the Presence of Butyric Acid

Candida albicans, Saccharomyces cerevisiae and their cell wall components, zymosan and glucan, have been shown to stimulate interleukin-8 (IL-8/CXCL-8) production by intestinal epithelial cell-like Caco-2 cells pre-cultured with 10 mM butyric acid. We examined in this study whether these yeasts also altered the production of other cytokines and cyclooxygenases (COXs) by Caco-2 cells. Culturing Caco-2 cells with 10 mM butyric acid and 15% FBS for 4 days enhanced the basal levels of mRNA encoding IL-6, IL-8, IL-18, monocyte chemoattractant protein (MCP)-1, stem cell factor, transforming growth factor (TGF)-β1, TGF-β3, tumor necrosis factor (TNF)-α, COX-1, and COX-2, but not of granulocyte-macrophage colony-stimulating factor (GM-CSF) and TGF-β2. The inclusion of live S. cerevisiae or C. albicans further enhanced the production of IL-8, but not of the other cytokines and COXs. The non-pathogenic yeasts, C. kefyr, C. utilis, C. versatilis, Kluyveromyces lactis, K. marxianus, Schizosaccharomyces pombe and Zygosaccharomyces rouxii, used for the production of fermented foods and probiotics, and the opportunistic pathogens, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis, isolated from human tissue samples also enhanced IL-8 secretion by Caco-2 cells.     

Uptake of 3,4-methylenedioxymethamphetamine and its related compounds by a proton-coupled transport system in Caco-2 cells

3,4-Methylenedioxymethamphetamine (MDMA) is an illegal amphetamine-type stimulant (ATS) that is abused orally in the form of tablets for recreational purposes. The aim of this work is to investigate the absorption mechanism of MDMA and other related compounds that often occur together in ATS tablets, and to determine whether such tablet components interact with each other in intestinal absorption. The characteristics of MDMA uptake by the human intestinal epithelial Caco-2 cell line were investigated. The Michaelis constant and the maximal uptake velocity at pH 6.0 were 1.11 mM and 13.79 nmol/min/mg protein, respectively, and the transport was electroneutral. The initial uptake rate was regulated by both intra- and extracellular pH. MDMA permeation from the apical to the basolateral side was inferior to that in the reverse direction, and a decrease in apical pH enhanced MDMA permeation from the basolateral to the apical side. These facts indicate that this transport system may be an antiporter of H⁺. However, under physiological conditions, the proton gradient cannot drive the MDMA uptake because it is inwardly directed. Large concentration differences of MDMA itself drive this antiporter. Various compounds with similar amine moieties inhibited the uptake, but substrates of organic cation transporters (OCT1-3) and an H⁺-coupled efflux antiporter, MATE, were not recognized.     

Inhibition of adhesion of Clostridium difficile to Caco-2 cells

Abstract For many microorganisms, including Clostridium difficile , mucosal association is an important factor influencing intestinal colonisation and subsequent infection. Inhibition of adhesion of C. difficile to intestinal mucosa could be a new promising strategy for prevention and treatment of antibiotic-associated diarrhoea. We investigated the possibilities of influencing the adhesion of C. difficile by xylitol and bovine colostrum.whey. Caco-2 cells and C. difficile cells were incubated with 1%, 5% and 10% solutions of xylitol and colostrum. Our study revealed that both xylitol and colostrum inhibited the adhesion of C. difficile to Caco-2 cells. Inhibition by xylitol was dose-dependent. When compared to the control, the count of adherent C. difficile decreased 3.4 times when treated with 1% xylitol, 12 times when 5% xylitol was applied, and 18.7 times when treated with 10% xylitol. The inhibition of adherence by colostrum was partially dose-dependent: 3.1 times in the case of 1%, and 5.5 times in the cases of 5% and 10% colostrum. Further experimental and clinical studies are needed for the application of xylitol and colostrum in the treatment and prophylaxis of pseudomembraneous colitis.     

Epithelial properties of human intestinal Caco-2 cells cultured in a serum-free medium

Human intestinal Caco-2 cells were cultured under serum-free conditions on an insoluble collagen and FCS matrix (Caco-2-SF), and a comparison was made between several characteristics of Caco-2 and Caco-2-SF cells. Their morphological appearance was identical. Slight differences were found in cell growth and expression of brush border enzymes between Caco-2 and Caco-2-SF cells. Similar levels of activity of Gly-Gly transport were expressed in both types of cell. Caco-2 cells cultured on permeable filters showed high transepithelial electrical resistance (TEER), indicating the high monolayer integrity. The transepithelial transport activity for glucose, alanine and Gly-Gly was detected by measuring the change in short-circuit current (Isc) after adding each of these nutrients to the apical chamber. In Caco-2-SF cells, such parameters as TEER and Isc were reduced drastically, suggesting that the monolayer integrity and cell polarity that are important for transepithelial transport were not attained. These parameters, however, could be restored by adding FCS or by milk whey. The result suggested that FCS and milk whey contain factors which regulate the formation of the tight junctions and, consequently, the development of cell polarity. Thus the Caco-2-SF cell-culture system will provide a useful model for studying factors which regulate the intestinal transepithelial transport functions.Abbreviations BCECF 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein - TEER transepithelial electrical resistance - LY lucifer yellow CH lithium salt     

Pharmacological levels of copper exert toxic effects in Caco-2 cells

Copper might be toxic to human intestinal cells because of its ability to catalyze the formation of free radicals. The aim of the present study was to quantify toxicological effects of increasing copper concentrations in preconfluent, colonic cancerous cells as well as in postconfluent, differentiating Caco-2 cells. Our results indicate that postconfluent cells might be more sensitive to copper toxicity. A significant rise of lactate dehydrogenase (LDH) release (150 μM or above) and decrease of cell proliferation (100 μM or above) with increasing copper levels was found, as compared to the control. To the contrary, preconfluent cells were not significantly affected by copper (LDH release) or, if so, only at a concentration of 250 μM (proliferation). Loss of viability and morphological changes, including loss of adherence and cell rounding, were visible after incubation with 250 μM copper in both groups. Superoxide dismutase (SOD) activities were not affected by copper. Glutathione peroxidase (GSH-Px) and catalase activities were higher in copper-treated cells, especially in the postconfluent ones (nevertheless, the results were not significant because of high standard deviations). In conclusion, we demonstrated that copper exerts intracellular, toxicological effects on both groups of Caco-2 cells, although the effects seem to be more evident in the postconfluent (enterocytelike) group. Risk assessment, especially for high concentrations, might be of special interest.     

氯化铬和吡啶羧酸铬在Caco-2细胞中的摄取和转运

采用一种体外培养的人小肠上皮细胞模型Caco-2研究了时间、浓度、温度对氯化铬和吡啶羧酸铬细胞摄取和跨细胞转运的影响。旨在探讨氯化铬和吡啶羧酸铬在小肠上皮细胞中的摄取和转运特点。结果表明:Caco-2细胞对氯化铬和吡啶羧酸铬的摄取和转运随浓度、时间而成线性增加,当温育温度从37℃降到4℃时,摄取和转运有下降趋势(P>0.05)。氯化铬和吡啶羧酸铬从肠腔侧(AP侧)到基底侧(BL侧)的表观通透系数(Papp)近似于BL侧到AP侧(分别为0.95～1.41倍和0.84～1.07倍)。氯化铬和吡啶羧酸铬的摄取率分别为(0.88±0.08)%和(4.73±0.60)%,转运率分别为(2.11±0.05)%和(9.08±0.25)%。结果提示氯化铬和吡啶羧酸铬是以被动扩散为主要方式被Caco-2细胞摄取和转运。     

Intestinal uptake of nateglinide by an intestinal fluorescein transporter

Nateglinide, a novel oral hypoglycemic agent, rapidly reaches its maximum serum concentration after oral administration, suggesting that it is rapidly absorbed in the intestine. However, nateglinide itself is not transported by MCT1 or PEPT1. The aim of this study was to characterize the transporters on the apical side of the small intestine that are responsible for the rapid absorption of nateglinide. It has been reported that the uptake of fluorescein by Caco-2 cells occurs via an H⁺-driven transporter and that the intestinal fluorescein transporter is probably not MCT1. We examined the contribution of the fluorescein transporter to the uptake of nateglinide by Caco-2 cells. Fluorescein competitively inhibited H⁺-dependent nateglinide uptake. All of fluorescein transporter inhibitors examined reduced the uptake of nateglinide. Furthermore, nateglinide inhibited fluorescein uptake. We conclude that the intestinal nateglinide/H⁺ cotransport system is identical to the intestinal fluorescein/H⁺ cotransport system.     

Structural Effects of Phenolic Acids on the Transepithelial Transport of Fluorescein in Caco-2 Cell Monolayers

The structural specificity of the monocarboxylic acid transporter (MCT) for the transport of phenolic acids was investigated by measuring the inhibitory effect on the fluorescein transport in Caco-2 cell monolayers. Although most of the monohydroxylated derivatives had an inhibitory effect, the di- and tri-hydroxylated ones did not. The methoxylated derivatives were more inhibitory than the hydroxylated ones in all the meta-substituted derivatives, suggesting that meta-hydroxylation of the substrate would decrease the affinity for MCT.