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 .     

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.     

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.     

Autocrine Production of Insulin-like Growth Factor-I (IGF-I) Affects Paracellular Transport Across Epithelial Cells In Vitro

Autocrine production of growth factors can have significant effects on cell activity. We report for the first time that autocrine production of insulin-like growth factor-I (IGF-I) alters paracellular transport across bovine mammary epithelial cells in vitro. Paracellular transport was assessed by measuring phenol red transport across mammary alveolar cells-large T antigen (MAC-T cells) derived from parental mammary epithelial cells, cultured on porous membranes and compared with two different transfected MAC-T cell lines that constitutively secrete IGF-I. Phenol red transport was essentially blocked in parental cell culture after six days, while IGF-I secreting cells provided essentially no barrier. Surprisingly, neither co-culture studies between parental and IGF-I-secreting cells nor addition of exogenous IGF-I or IGF-binding protein-3 reversed the phenol red transport properties. IGF-I-secreting cells did however express lower levels of the junction components occludin and E-cadherin than parental cells, suggesting that localized autocrine IGF-I activity might lead to increased permeability via changes in both the tight and adherens junction protein levels.     

Caco-2 Cell Line Used as an In Vitro Model to Study Cadmium Accumulation in Intestinal Epithelial Cells

¹⁰⁹Cd uptake was studied using the highly differentiated TC7 clone of Caco-2 cells as a model of human enterocyte function. Intracellular accumulation of 0.3 μm ¹⁰⁹Cd involved a rapid and a slow uptake phase, which resulted in complete equilibration (t _?= 17.3 ± 1.3 min) with an apparent in-to-out distribution ratio (α _e ) of 11.6 ± 0.8. The amplitude of the rapid phase (U ₀) and the rate of the slow phase (V) were similarly reduced in the less differentiated PF11 clone, but comparable α _e values were observed at equilibrium. In both clones, the t _? and α _e values increased and decreased, respectively, upon addition of unlabeled Cd to the uptake media. In TC7 cells, ¹⁰⁹Cd uptake at 1 min (U ₁) was unaffected by Ca concentrations four order of magnitude in excess, but both U ₀ and V demonstrated similar sensitivities to unlabeled Cd, Zn and sulfhydryl-reactive agents. Only U ₀ disappeared when EDTA was present in the wash solutions. U ₁ showed saturation kinetics and the data were found compatible with a model assuming rapid initial Cd binding and transport through a unique transport protein (K _m = 3.8 ± 0.7 μm). Cd efflux kinetics demonstrated partial reversibility in EDTA-containing solutions, suggesting that the taken up Cd might be both tightly and loosely bound to intracellular binding sites. However, the displacement of ¹⁰⁹Cd measured at 65 min failed to reveal this heterogeneity: the data were found compatible with a model equation assuming the presence of one class of high-capacity high-affinity binding sites. We conclude that a slow-transport fast-intracellular binding mechanism of Cd uptake best accounts for these results and that Cd transport most likely involves a carrier-type of protein unrelated to Ca absorption. Received: 19 January 1996/Revised: 23 January 1997     

<Emphasis Type="Italic">In vitro</Emphasis> Permeability Enhancement in Intestinal Epithelial Cells (Caco-2) Monolayer of Water Soluble Quaternary Ammonium Chitosan Derivatives

The aim of this study was to investigate the effects of a type of hydrophobic moiety, extent of N-substitution (ES), and degree of quaternization (DQ) of chitosan (CS) on the transepithelial electrical resistance and permeability of Caco-2 cells monolayer, using fluorescein isothiocyanate dextran 4,400 (FD-4) as the model compound for paracellular tight junction transport. CS was substituted with hydrophobic moiety, an aliphatic aldehyde (n-octyl) or aromatic aldehyde (benzyl), for the improved hydrophobic interaction with cell membrane, and they were quaternized with Quat-188 to render CS soluble. The factors affecting the epithelial permeability have been evaluated in the intestinal cell monolayers, Caco-2 cells. Cytotoxicity was evaluated by using the trypan blue and MTT viability assay. The results revealed that at pH 7.4 CSQ appeared to increase cell permeability in dose-dependent manner, and this effect was relatively reversible at the lower doses of 0.05–1.25 mM. The higher DQ and ES caused the higher permeability of FD-4. Cytotoxicity of CSQ was concentration, %DQ, and %ES dependent. Substitution with hydrophobic moiety caused decreasing in permeability of FD-4 and cytotoxicity by benzyl group had more effect than octyl group. These studies demonstrated that these novel modified chitosan derivatives had potential for using as absorption enhancers.     

Multiple effects of mercury on cell volume regulation, plasma membrane permeability, and thiol content in the human intestinal cell line Caco-2

In a previous study, we characterized Cd–Hg interactions for uptake in human intestinal Caco-2 cells. We pursued our investigations on metal uptake from metal mixtures, focusing on the effects of Hg on cellular homeostasis. A 4-fold higher equilibrium accumulation value of 0.3 μmol/L ²⁰³Hg was measured in the presence of 100 μmol/L unlabeled Hg in the serum-free exposure medium without modification in the initial uptake rate. This phenomenon was eliminated at 4^∘C. Mercury induced an increase in tritiated water and [³H]mannitol uptakes for exposure times greater than 20 min. Incubations for 20 min and 30 min with 100 μmol/L Hg and 2 mmol/L N-ethylmaleimide (NEM) resulted in a 34% and 50% reductions in cellular thiol staining, respectively, with additive effects. Lactate dehydrogenase leakage and live/dead assays confirmed the maintenance of cell membrane integrity in Hg- or NEM-treated cells. We conclude that Hg may alter membrane permeability and increase cell volume without any loss in cell viability. This phenomenon is sensitive to temperature and could involve Hg interaction with membrane thiols, possibly related to solute transport. During metal uptake from metal mixtures, Hg may thus promote the uptake of other toxic metals by increasing cell volume and consequently cell capacity. Deceased 25 March 2004     

A Link between FXYD3 (Mat-8)-mediated Na,K-ATPase Regulation and Differentiation of Caco-2 Intestinal Epithelial Cells

FXYD3 (Mat-8) proteins are regulators of Na,K-ATPase. In normal tissue, FXYD3 is mainly expressed in stomach and colon, but it is also overexpressed in cancer cells, suggesting a role in tumorogenesis. We show that FXYD3 silencing has no effect on cell proliferation but promotes cell apoptosis and prevents cell differentiation of human colon adenocarcinoma cells (Caco-2), which is reflected by a reduction in alkaline phosphatase and villin expression, a change in several other differentiation markers, and a decrease in transepithelial resistance. Inhibition of cell differentiation in FXYD3-deficient cells is accompanied by an increase in the apparent Na⁺ and K⁺ affinities of Na,K-ATPase, reflecting the absence of Na,K-pump regulation by FXYD3. In addition, we observe a decrease in the maximal Na,K-ATPase activity due to a decrease in its turnover number, which correlates with a change in Na,K-ATPase isozyme expression that is characteristic of cancer cells. Overall, our results suggest an important role of FXYD3 in cell differentiation of Caco-2 cells. One possibility is that FXYD3 silencing prevents proper regulation of Na,K-ATPase, which leads to perturbation of cellular Na⁺ and K⁺ homeostasis and changes in the expression of Na,K-ATPase isozymes, whose functional properties are incompatible with Caco-2 cell differentiation.     

Cadmium accumulation and interactions with zinc, copper, and manganese, analysed by ICP-MS in a long-term Caco-2 TC7 cell model

The influence of long-term exposure to cadmium (Cd) on essential minerals was investigated using a Caco-2 TC7 cells and a multi-analytical tool: microwave digestion and inductively coupled plasma mass spectrometry. Intracellular levels, effects on cadmium accumulation, distribution, and reference concentration ranges of the following elements were determined: Na, Mg, Ca, Cr, Fe, Mn, Co, Ni, Cu, Zn, Mo, and Cd. Results showed that Caco-2 TC7 cells incubated long-term with cadmium concentrations ranging from 0 to 10 μmol Cd/l for 5 weeks exhibited a significant increase in cadmium accumulation. Furthermore, this accumulation was more marked in cells exposed long-term to cadmium compared with controls, and that this exposure resulted in a significant accumulation of copper and zinc but not of the other elements measured. Interactions of Cd with three elements: zinc, copper, and manganese were particularly studied. Exposed to 30 μmol/l of the element, manganese showed the highest inhibition and copper the lowest on cadmium intracellular accumulation but Zn, Cu, and Mn behave differently in terms of their mutual competition with Cd. Indeed, increasing cadmium in the culture medium resulted in a gradual and significant increase in the accumulation of zinc. There was a significant decrease in manganese from 5 μmol Cd/l exposure, and no variation was observed with copper.     

The Effect of Linker Length on the Photomodification of Tyr Residue in N-(Tyrosyl)-N"-(5-azido-2-nitrobenzoyl)-diaminoalkanes

N-(Tyrosyl)-N"-(5-azido-2-nitrobenzoyl)-1,4-diaminobutane containing a Tyr residue connected with the photoreactive aryl azide group through a diaminobutylene linker was synthesized as a model for studying the photomodification of Tyr residues in proteins. This compound and the compound with a shorter, 1,2-diaminoethylene linker, obtained previously, were subjected to a computer modeling to find their minimal energy conformations. The aromatic rings of Tyr and 5-azido-2-nitrobenzoic acid residues in the latter compound were localized in parallel planes at a distance of approximately 0.3 nm between them and were shown to be implicated in stacking interaction. On the contrary, the planes of aromatic rings of the former compound with a longer diaminobutylene linker were found to be situated perpendicularly to each other, with the distance between the centers of the rings being approximately 0.6 nm. The computer analysis was confirmed by experimental results: when studying the photomodification of the compound with the diaminobutylene linker, neither stable products of the Tyr photomodification nor unstable products capable of transformation into stable products in the dark were found. On the contrary, such products were previously identified in the case of the compound with diaminoethylene linker. The formation of amino, nitro, azoxy and azo derivatives was common for the photomodification of both compounds.     

Synthesis and in vitro anti-HIV activity of N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-4-yl)acetamide derivatives using MTT method

A series of novel N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-4-yl)acetamide derivatives has been synthesized. All the newly synthesized compounds were evaluated for their anti-HIV activity using MTT method. Most of these compounds showed moderate to potent activity against wild-type HIV-1 with an EC₅₀ ranging from >7 EC₅₀ [μg/ml] to <100 EC₅₀ [μg/ml]. Among them, N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-4-yl)acetamide 6v was identified as the most promising compound (EC₅₀ = <7 μg/ml). Among all the compounds, three compounds 6m, 6v and 6u have been exhibits potent anti-HIV activity against MT-4 cells.     

Regulation of Intestinal Epithelial Brush Border Na+/H+ Exchanger Isoforms, NHE2 and NHE3, in C2bbe Cells

Until recently, studies to characterize the intestinal epithelial Na⁺/H⁺ exchangers had to be done in nonepithelial, mutated fibroblasts. In these cells, detection of any Na⁺/H⁺ exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to measure NHE activity. Because changes in pH _i only approximate Na⁺/H⁺ exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation, we have used ²²[Na] unidirectional apical to cell uptake to measure activities specific to NHE2 or NHE3. Furthermore, we performed these measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental precondition. Both brush border NHEs, when expressed in the well-differentiated, intestinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical domain and are regulated by second messengers in the same way they are regulated in vivo. Increases in intracellular calcium and cAMP inhibit both isoforms, while phorbol ester affects only NHE3. NHE2 inhibition by cAMP and Ca⁺⁺ involves changes to both K _Na and V _max . In contrast, the same two second messengers inhibit NHE3 by a decrease in V _max exclusively. Phorbol ester activation of protein kinase C alters both V _max and K _Na of NHE3, suggesting a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in epithelial cells, are basally active and are differentially regulated by signal transduction pathways. Received: 28 January 1999/Revised: 18 May 1999     

Biological activities and structure-activity relationship of substitution compounds of N-[2-(3-indolyl)ethyl]succinamic acid and N-[2-(1-naphthyl)ethyl]succinamic acid, derived from a new category of root-promoting substances, N-(phenethyl)succinamic acid analogs

In a previous study, it was demonstrated that N-(phenethyl)succinamic acid (PESA) derivatives form a new category of root-promoting substances which do not exhibit auxin-like activities, such as stem elongation and leaf epinasty (Soejima et al., 2000 [Plant Cell Physiol. 41s: 197]). In this study, N-[2-(3-indolyl)ethyl]succinamic acid (IESA) and N-[2-(1-naphthyl)ethyl]succinamic acid (NESA) were synthesized, and their biological activities were evaluated. In an adzuki root-promoting assay, IESA and NESA exhibited root-promoting activity equivalent to PESA. In adzuki stem elongation assays, elongation activity was not observed in the stem segments soaked in either an IESA or NESA aqueous solution, whereas the stem segments immersed in Indole-3-acetic acid (IAA) or 1-naphthylacetic acid (NAA) aqueous solution were clearly elongated. In an epinastic bending study, IAA and NAA exhibited leaf epinasty, whereas IESA and NESA did not, suggesting that the IESA and NESA derivatives belong to the same category of root-promoting substances as PESA derivatives and are different from auxin-like substances. In addition, eleven kinds of IESA derivatives and nineteen kinds of NESA derivatives were synthesized, and their root-promoting activities were measured. The activities of methyl ester derivatives were approximately three times higher than that of the acid compounds, with exceptions for some compounds. The partition coefficient (P) between 1-octanol and water for each IESA, NESA, and PESA derivative was measured in order to evaluate the hydrophobicity of their molecules and to determine their structure–activity relationship. The results indicate that the root-promoting activity of the acid compounds was significantly correlated with their hydrophobicity, whereas that of ester derivatives was not correlated.     

N-(2-Carboxyethyl)chitosans: regioselective synthesis,characterisation and protolytic equilibria

N-(2-Carboxyethyl)chitosans were obtained by reaction of low molecular weight chitosan with a low degree of acetylation and 3-halopropionic acids under mild alkaline media (pH 8-9, NaHCO3) at 60 degrees C. The chemical structure of the derivatives obtained was determined by 1H and 13C NMR spectroscopies. It was found that alkylation of chitosan by 3-halopropionic acids proceeds exclusively at the amino groups. The products obtained are described in terms of their degrees of carboxyethylation and ratio of mono-, di-substitution and free amine content. The protonation constants of amino and carboxylate groups of a series of N-(2-carboxyethyl)chitosans were determined by pH-titration at ionic strength 0.1 M KNO3 and 25 degrees C.     

Econazole-evoked [Ca2+]i Rise and Non-Ca2+-triggered Cell Death in Rabbit Corneal Epithelial Cells (SIRC)

The effects of econazole, an antifungal drug applied for treatment of keratitis and mycotic corneal ulcer, on cytosolic-free Ca²⁺ concentrations ([Ca²⁺]_i) and viability of corneal cells was examined by using SIRC rabbit corneal epithelial cells as model. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations ≥ 1 µM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The econazole-induced Ca(2+) influx was insensitive to L-type Ca²⁺ channel blockers and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 20 µM econazole, [Ca²⁺]_i rises induced by 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) were abolished. Conversely, thapsigargin pretreatment also abolished econazole-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 µM U73122 did not change econazole-induced [Ca²⁺]_i rises. At concentrations between 10 and 80 µM, econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 20 µM econazole was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. This shows that in SIRC cells econazole induces [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from unknown pathways. Econazole-caused cytotoxicity was independent from a preceding [Ca²⁺]_i rise.     

1-N-methyl-(6E)-(2-methylpropylidene)-(3Z)-3-(phenylmethylene)-2,5-piperazinedione,a metabolite from Streptomyces albus

The structure and stereochemistry of a new piperazinedione, isolated from the cells of Streptomyces albus, are assigned on the basis of spectroscopic data, including comparison with related 2,5-piperazinediones.     

C3d and Epstein-Barr Virus (CR2/CD21 Ligands) Stimulate Cells of an HTLV-I Line,MT-2

We studied the physiological role of complement receptor type II (CR2, C3d/EBV receptor) expressed on T cells using MT-2 cells. First, we confirmed CR2 expression on MT-2 cells by flow cytometry and found that the MW of CR2 molecules on these cells and Raji B cells were the same by SDS-PAGE analysis. When MT-2 lysates were incubated with anti-CR2 mAb HB5 and thereafter with ³²P-labeled ATP, 52- and 74-kDa proteins were phosphorylated, suggesting the activation of MT-2 cells through the complex of CR2 with these proteins. In this respect, we measured lymphotoxin production by MT-2 cells when incubated with C3d or EBV. The cytotoxicity of the MT-2 supernatant against L929 cells was elevated in a dose- and time-dependent manner. Next, we confirmed EBNA expression on EBV-infected MT-2 cells and attempted to establish an EBV-positive MT-2 clone by in vitro EBV infection. However, these clones disappeared during cloning. To clarify this mechanism, we examined the EBV genome in MT-2 cells. By Southern blot analysis, BamHI digestion of DNA extracts from MT-2 cells 3 days after EBV treatment gave a 3.0-kb signal which comigrated with the EBV BamHI-W probe. The 3.0-kb signal of genomic EBV-DNA was detected at 1, 2, 3, 5, and 7 days after EBV treatment, but could not be detected at 14 days. Thus, natural ligands of CR2 stimulate CR2-positive MT-2 cells through their functionally active CR2 molecules and in vitro EBV infection of MT-2 cells might be transient.     

Identification of a Novel Lipofuscin Pigment (iisoA2E) in Retina and Its Effects in the Retinal Pigment Epithelial Cells

Lipofuscin accumulation in retinal pigment epithelial (RPE) cells of the eye implicates the etiologies of Stargardt disease and age-related macular degeneration, a leading cause of blindness in the elderly. Here, we have identified a previously unknown RPE lipofuscin component. By one- and two-dimensional NMR techniques and mass spectrometry, we confirmed that this compound is a new type of pyridinium bisretinoid presenting an unusual structure, in which two polyenic side chains are attached to adjacent carbons of a pyridinium ring. This pigment is a light-induced isomer of isoA2E, rather than A2E, referred to as iisoA2E. This pigment is a fluorescent lipofuscin compound with absorbance maxima at ∼430 and 352 nm detected in human, pig, mouse, and bovine eyes. Formation of iisoA2E was found in reaction mixtures of all-trans-retinal and ethanolamine. Excess intracellular accumulation of this adduct in RPE cells in vitro leads to a significant loss of cell viability and caused membrane damage. Phospholipase D-mediated phosphodiester cleavage of the A2PE series generated isoA2E and iisoA2E, in addition to A2E, thus corroborating the presence of isoA2PE and iisoA2PE that may serve as biosynthetic precursors of isoA2E and iisoA2E.