Human Intestinal Cell Monolayers Research Articles

Colon-available mango (poly)phenols exhibit mitigating effects on the intestinal barrier function in human intestinal cell monolayers under inflammatory conditions.

This study investigated the impact of in vivo available colon-mango (poly)phenols on stress-induced impairment of intestinal barrier function. Caco-2/HT29-MTX cells were incubated with six extracts of ileal fluid collected pre- and 4-8 h post-mango consumption before being subjected to inflammatory stress. (Poly)phenols in ileal fluids were analysed by UHPLC-HR-MS. Epithelial barrier function was monitored by measurement of trans-epithelial electrical resistance (TEER) and the production of selected inflammatory markers (interleukin-8 (IL-8) and nitric oxide (NO)) and the major mucin of the mucosal layer (MUC2). Post-mango intake ileal fluids contained principally benzoic acids, hydroxybenzenes and galloyl derivatives. There was a high interindividual variability in the levels of these compounds, which was reflected by the degree of variability in the protective effects of individual ileal extracts on inflammatory changes in the treated cell cultures. The 24 h treatment with non-cytotoxic doses of extracts of 4-8 h post-mango intake ileal fluid significantly reduced the TEER decrease in monolayers treated with the inflammatory cytomix. This effect was not associated with changes in IL-8 expression and secretion or claudine-7 expression. The mango derived-ileal fluid extract (IFE) also mitigated cytomix-dependent nitrite secretion, as a proxy of NO production, and the MUC2 reduction observed upon the inflammatory challenge. These insights shed light on the potential protective effect of mango (poly)phenols on the intestinal barrier exposed to inflammatory conditions.

Enterotoxic potential of dietary cholesterol autoxidation products

Background In both Western and Westernized diets, together with a relatively high amount of cholesterol, variable amountsof its oxidized metabolites, oxysterols, are consistently consumed. These oxysterols, mostly of non-enzymatic origin, are produced during sterol autoxidation in foodstuff manufacturing and storage. Objective This study aimed to analyze the potential enterotoxic effects of all main oxysterols of non-enzymatic origin so far identified in a variety of foods. Experimental plan Differentiated human intestinal cell monolayers (CaCo-2) were incubated up to 48 h in the presence or absence of 0.5, 1 or 5 µM with one out of seven non-enzymatic oxysterols, prior to the verification of minimal irreversible cell damage within the chosen concentration range. Results All tested oxysterols were proven to exert damaging effects on cell monolayers in vitro. The inflammatory interleukin-8 and monocyte chemotactic protein-1 were mostly upregulated by 7-ketocholesterol and 7β-hydroxycholesterol, respectively, then to a lower extent by 5α,6α-epoxycholesterol, 7α-hydroxycholesterol and 5β,6β-epoxycholesterol. 7-Ketocholesterol and 7β-hydroxycholesterol also appeared to be most effective in impairing claudin-1, occludin and E-cadherin proteins, followed by 25-hydroxycholesterol and triol. Conclusions The oxysterols consistently derived by food autoxidation were tested; they potentially impaired the integrity of the intestinal epithelial barrier and triggered an inflammatory response within 0.5–5 µM concentrations, easily reachable in a single Western meal. Significance Statement This comprehensive analysis focused on the potential impairment of the intestinal barrier by the main dietary non-enzymatic oxysterols, should guide further nutrition research aiming at defining a threshold amount of these cholesterol derivatives in order not to derange the physiological gut–brain axis.

In vitro anti-hyperglycemic, antioxidant activities and intestinal glucose uptake evaluation of Endiandra kingiana extracts

Abstract Optimal control of postprandial hyperglycemia is essentially important in the management of diabetes mellitus. The present investigation was undertaken to evaluate the in vitro anti-hyperglycemic, antioxidant properties and intestinal glucose uptake inhibition of Endiandra kingiana (E. kingiana) extracts. Previously, our group has identified and characterized the bioactive compounds of E. kingiana extracts, which had discovered several polyketides; endiandric acids and kingianins. Here, the inhibitory potential of bark-ethyl acetate (BEA), bark-methanol (BM), leaf-ethyl acetate (LEA), and leaf-methanol (LM) extracts of E. kingiana against carbohydrate-hydrolyzing enzymes with their mode of inhibitions were evaluated. Further, the antioxidant activities and inhibitory potential on glucose uptake in Caco-2 human intestinal cell monolayers were determined. Our finding showed that BEA extract exhibited the most potent inhibition activities against α-amylase (IC50 = 2.32 μg/mL) and α-glucosidase (IC50 = 1.83 μg/mL) by following inhibition mode of competitive and non-competitive manners, respectively. Meanwhile, BM extract exhibited notable antioxidant capabilities, as evidenced by strongest free radical scavenging (IC50 = 1.18 μg/mL) and reducing power effect (118.53 mM Fe2+ equivalent/g extract) in couple with highest total phenolic contents (10.17 mg GAE/g extract) compared to other extracts. Mechanistically, both BEA and BM extracts of E. kingiana significantly inhibited glucose uptake in Caco-2 cell monolayers under sodium-dependent condition. Collectively, these findings suggest that BM and BEA extracts of E. kingiana exert in vitro anti-hyperglycemic and antioxidant properties, which can be further utilized as a potential candidate for treatment of hyperglycemia–induced oxidative stress conditions.

Catechin and Procyanidin B2 Modulate the Expression of Tight Junction Proteins but Do Not Protect from Inflammation-Induced Changes in Permeability in Human Intestinal Cell Monolayers.

The possibility of counteracting inflammation-related barrier defects with dietary compounds such as (poly)phenols has raised much interest, but information is still scarce. We have investigated here if (+)-catechin (CAT) and procyanidin B2 (PB2), two main dietary polyphenols, protect the barrier function of intestinal cells undergoing inflammatory stress. The cell model adopted consisted of co-cultured Caco-2 and HT29-MTX cells, while inflammatory conditions were mimicked through the incubation of epithelial cells with the conditioned medium of activated macrophages (MCM). The epithelial barrier function was monitored through trans-epithelial electrical resistance (TEER), and ROS production was assessed with dichlorofluorescein, while the expression of tight-junctional proteins and signal transduction pathways were evaluated with Western blot. The results indicated that MCM produced significant oxidative stress, the activation of NF-κB and MAPK pathways, a decrease in occludin and ZO-1 expression, and an increase in claudin-7 (CL-7) expression, while TEER was markedly lowered. Neither CAT nor PB2 prevented oxidative stress, transduction pathways activation, ZO-1 suppression, or TEER decrease. However, PB2 prevented the decrease in occludin expression and both polyphenols produced a huge increase in CL-7 abundance. It is concluded that, under the conditions adopted, CAT and PB2 do not prevent inflammation-dependent impairment of the epithelial barrier function of intestinal cell monolayers. However, the two compounds modify the expression of tight-junctional proteins and, in particular, markedly increase the expression of CL-7. These insights add to a better understanding of the potential biological activity of these major dietary flavan-3-ols at intestinal level.

3,3'-Diindolylmethane Improves Intestinal Permeability Dysfunction in Cultured Human Intestinal Cells and the Model Animal Caenorhabditis elegans.

3,3'-Diindolylmethane (DIM), a digestive metabolite originating from cruciferous vegetables, has dietary potential for the treatment of various human intestinal diseases. Although intestinal permeability dysfunction is closely related to the initiation and progression of human intestinal inflammatory diseases (IBDs), the effect of DIM on intestinal permeability is unclear. We evaluated the effect of DIM on the intestinal permeability of human intestinal cell monolayers and the animal model Caenorhabditis elegans, which were treated with IL-1β and Pseudomonas aeruginosa, respectively, to mimic IBD conditions. DIM substantially restored the intestinal permeability of differentiated Caco-2 cells by enhancing the expression of tight junction proteins (including occludin and ZO-1). Compared to the IL-1β single treatment (551.0 ± 49.0 Ω·cm2), DIM (10 μM) significantly increased the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers (919.0 ± 66.4 Ω·cm2, p < 0.001). DIM also ameliorated the impaired intestinal permeability and extended the lifespan of C. elegans fed P. aeruginosa. The mean lifespan of DIM-treated worms (10.8 ± 1.3 days) was higher than that of control-treated worms (9.7 ± 1.1 days, p < 0.01). Thus, DIM is a potential nutraceutical candidate for the treatment of leaky gut syndrome by improving intestinal permeability.

Potato phenolics impact starch digestion and glucose transport in model systems but translation to phenolic rich potato chips results in only modest modification of glycemic response in humans

Beneficial effects of some phenolic compounds in modulation of carbohydrate digestion and glycemic response have been reported, however effects of phenolics from processed potato products on these endpoints are not well known. The aims of this study were to characterize phenolic profiles of fresh potatoes (purple, red, or white fleshed; 2 varieties each) and chips, and to examine the potential for potato phenolic extracts (PPE) to modulate starch digestion and intestinal glucose transport in model systems. Following in vitro assessment, a pilot clinical study (n=11) assessed differences in glycemic response and gastric emptying between chips from pigmented and white potatoes. We hypothesized that phenolics from pigmented potato chips would be recovered through processing and result in a reduced acute glycemic response in humans relative to chips made from white potatoes. PPEs were rich in anthocyanins (~98, 11 and ND mg/100 g dw) and chlorogenic acids (~519, 425 and 157 mg/100 g dw) for purple, red and white varieties respectively. While no significant effects were observed on starch digestion by α-amylase and the α-glucosidases, PPEs significantly (p<0.05) decreased the rate of glucose transport, measured following transport of 1,2,3,4,5,6,6-d7 -glucose (d7-glu) across Caco-2 human intestinal cell monolayers, by 4.5-83.9%. Consistent with in vitro results, consumption of purple potato chips modestly but significantly (p<0.05) decreased blood glucose at 30 and 60 minutes post consumption compared to white chips without impacting gastric emptying. These results suggest that potato phenolics may play a modest role in modulation of glycemic response and these effects may result in subtle differences between consumer products.

Stability and absorption of anthocyanins from blueberries subjected to a simulated digestion process

Numerous studies have shown that anthocyanins usually have better in vitro bioactivity than in vivo bioactivity. This may be due to physiochemical degradation during gastrointestinal digestion and their poor bioavailability in in vivo studies. Therefore, this study aims to investigate the effects of anthocyanin structure on their stability under simulated gastrointestinal digestion and to assess their absorption in the intestines using Caco-2 human intestinal cell monolayers. The results show that gastric digestion does not significant affect blueberry anthocyanins in terms of composition and antioxidative activity. However, approximately 42% of the total anthocyanin and 29% of the antioxidative activity were lost during intestinal digestion. Structural analysis indicated that fewer free hydroxyl groups and more methoxy groups in the B-ring improve anthocyanin stability. The absorption trials demonstrated that more hydrophobic anthocyanins have better absorption efficiency than more hydrophilic anthocyanins. Furthermore, the glycoside structure also determines the absorption efficiency of anthocyanins.

Bioactive Dietary Polyphenolic Compounds Reduce Nonheme Iron Transport across Human Intestinal Cell Monolayers

There is persuasive epidemiological evidence that regular intake of dietary bioactive polyphenolic compounds promotes human health. Because dietary polyphenolic compounds have a wide range of effects in vivo and vitro, including chelation of metals such as iron, it is prudent to test whether the regular consumption of bioactive polyphenolic components impair the utilization of dietary iron. We examined the influence of the dietary polyphenols (-) -epigallocatechin-3-gallate (EGCG) and grape seed extract (GSE) on transepithelial iron transport in Caco-2 intestinal cells. The range of EGCG and GSE concentrations used in this study was within physiological levels and did not affect the integrity of differentiated Caco-2 cell monolayers. Both EGCG and GSE decreased (P < 0.001) transepithelial iron transport. However, apical iron uptake was increased (P < 0.001) by the addition of EGCG and GSE. The increased uptake of iron might be due in part to the reducing activity of EGCG and GSE. Both EGCG and GSE reduced ∼15% of the applied Fe3+ to Fe2+ in the uptake buffer. Despite the increased cellular levels of 55Fe, the transfer of iron across the basolateral membrane of the enterocyte was extremely low, indicating that basolateral exit via ferroportin-1 was impaired, possibly through formation of a nontransportable polyphenol-iron complex. Our data show that polyphenols inhibit nonheme iron absorption by reducing basolateral iron exit rather than by decreasing apical iron import in intestinal cells.

Absorption of Anthocyanins from Blueberry Extracts by Caco-2 Human Intestinal Cell Monolayers

Recent studies have shown that dietary polyphenols may contribute to the prevention of cardiovascular disease and cancer. Anthocyanins from different plant sources including blueberries have been shown to possess potential anticancer activities. One of the key factors needed to correctly relate the in vitro study results to human disease outcomes is information about bioavailability. The objectives of the current study were to evaluate the absorption of blueberry anthocyanin extracts using Caco-2 human intestinal cell monolayers and investigate the effects of different aglycones, sugar moieties, and chemical structure on bioavailability of different types of anthocyanins. The results of this study showed that anthocyanins from blueberries could be transported through the Caco-2 cell monolayers although the transport/absorption efficiency was relatively low compared to other aglycone polyphenols. The transport efficiency of anthocyanins averaged approximately 3-4% [less than 1% in delphinidin glucoside (Dp-glc)]. No significant difference in transport/absorption efficiency was observed among three blueberry cultivars. The observed trends among different anthocyanins generally agreed well with some published in vivo results. Dp-glc showed the lowest transport/absorption efficiency, and malvidin glucoside (Mv-glc) showed the highest transport/absorption efficiency. Our result indicates that more free hydroxyl groups and less OCH(3) groups can decrease the bioavailability of anthocyanins. In addition, cyanindin glucoside (Cy-glc) showed significantly higher transport efficiency than cyanidin galactoside (Cy-gal), and peonidin glucoside (Pn-glc) showed significantly higher transport efficiency than peonidin galactoside (Pn-gal), indicating that glucose-based anthocyanins have higher bioavailability than galactose-based anthocyanins.

Aeromonas hydrophila beta-hemolysin induces active chloride secretion in colon epithelial cells (HT-29/B6).

The diarrheal mechanisms in Aeromonas enteritis are not completely understood. In this study we investigated the effect of aeromonads and of their secretory products on ion secretion and barrier function of monolayers of human intestinal cells (HT-29/B6). Ion secretion was determined as a short-circuit current (I(SC)) of HT-29/B6 monolayers mounted in Ussing-type chambers. Transepithelial resistance (R(t)) served as a measure of permeability. A diarrheal strain of Aeromonas hydrophila (strain Sb) added to the mucosal side of HT-29/B6 monolayers induced a significant I(SC) (39 +/- 3 microA/cm(2)) and decreased the R(t) to approximately 10% of the initial value. A qualitatively identical response was obtained with sterile supernatant of strain Sb, and Aeromonas supernatant also induced a significant I(SC) in totally stripped human colon. Tracer flux and ion replacement studies revealed the I(SC) to be mainly accounted for by electrogenic Cl(-) secretion. Supernatant applied serosally completely abolished basal I(SC). The supernatant-induced I(SC) was inhibited by the protein kinase C inhibitor chelerythrine, whereas a protein kinase A inhibitor (H8) and a Ca(2+) chelator (BAPTA-AM) had no effect. Physicochemical properties indicated that the supernatant's active compound was an aerolysin-related Aeromonas beta-hemolysin. Accordingly, identical I(SC) and R(t) responses were obtained with Escherichia coli lysates harboring the cloned beta-hemolysin gene from strain SB or the aerA gene encoding for aerolysin. Sequence comparison revealed a 64% homology between aerolysin and the beta-hemolysin cloned from Aeromonas sp. strain Sb. In conclusion, beta-hemolysin secreted by pathogenic aeromonads induces active Cl(-) secretion in the intestinal epithelium, possibly by channel insertion into the apical membrane and by activation of protein kinase C.

Diarrhea-associated HIV-1 APIs potentiate muscarinic activation of Cl- secretion by T84 cells via prolongation of cytosolic Ca2+ signaling.

Aspartyl protease inhibitors (APIs) effectively extend the length and quality of life in human immunodeficiency virus (HIV)-infected patients, but dose-limiting side effects such as lipodystrophy, insulin resistance, and diarrhea have limited their clinical utility. Here, we show that the API nelfinavir induces a secretory form of diarrhea in HIV-infected patients. In vitro studies demonstrate that nelfinavir potentiates muscarinic stimulation of Cl(-) secretion by T84 human intestinal cell monolayers through amplification and prolongation of an apical membrane Ca(2+)-dependent Cl(-) conductance. This stimulated ion secretion is associated with increased magnitude and duration of muscarinically induced intracellular Ca(2+) transients via activation of a long-lived, store-operated Ca(2+) entry pathway. The enhanced intracellular Ca(2+) signal is associated with uncoupling of the Cl(-) conductance from downregulatory intracellular mediators generated normally by muscarinic activation. These data show that APIs modulate Ca(2+) signaling in secretory epithelial cells and identify a novel target for treatment of clinically important API side effects.

Hormonal regulation of dipeptide transporter (PepT1) in Caco-2 cells with normal and anoxia/reoxygenation management.

To determine the regulation effects of recombinant human growth hormone (rhGH) on dipeptide transporter(PepT1) in Caco-2 cells with normal culture and anoxia/reoxygenation injury. A human intestinal cell monolayer (Caco-2) was used as the in vitro model of human small intestine and cephalexin as the model substrate for dipeptide transporter (PepT1). Caco-2 cells grown on Transwell membrane filters were preincubated in the presence of rhGH in the culture medium for 4 d, serum was withdrawn from monolayers for 24 h before each experiment. The transport experiments of cephalexin across apical membromes were then conducted; Caco-2 cells grown on multiple well dishes (24 pore) with normal culture or anoxia/reoxygenation injury were preincubated with rhGH as above and uptake of cephalexin was then measured. The transport and uptake of cephelaxin across apical membranes of Caco-2 cells after preincubation with rhGH were significantly increased compared with controls (P=0.045, 0.0223). Also, addition of rhGH at physiological concentration (34 nM) to incubation medium greatly stimulates cephalexin uptake by anoxia/reoxygenation injuried Caco-2 cells (P=0.0116), while the biological functions of PepT1 in injured Caco-2 cells without rhGH were markedly downregulated. Northern blot analysis showed that the level of PepT1 mRNA of rhGH-treated injured Caco-2 cells was greatly increased compared to controls. The present results of rhGH stimulating the uptake and transport of cephalexin indicated that rhGH greatly upregulates the physiological effects of dipeptide transporters of Caco-2 cells. The alteration in the gene expression may be a mechanism of regulation of PepT1. In addition, Caco-2 cells take up cephalexin by the Proton-dependent dipeptide transporters that closely resembles the transporters present in the intestine. Caco-2 cells represent an ideal cellular model for future studies of the dipeptide transporter.

Methionine oxidation as a major cause of the functional impairment of oxidized actin

A significant specific increase in the actin carbonyl content has been recently demonstrated in human brain regions severely affected by the Alzheimer’s disease pathology, in postischemic isolated rat hearts, and in human intestinal cell monolayers following incubation with hypochlorous acid (HOCl). We have very recently shown that exposure of actin to HOCl results in the immediate loss of Cys-374 thiol, oxidation of some methionine residues, and, at higher molar ratios of oxidant to protein, increase in protein carbonyl groups, associated with filament disruption and inhibition of filament formation. In the present work, we have studied the effect of methionine oxidation induced by chloramine-T (CT), which at neutral or slightly alkaline pH oxidizes preferentially Met and Cys residues, on actin filament formation and stability utilizing actin blocked at Cys-374. Methionines at positions 44, 47, and 355, which are the most solvent-exposed methionyl residues in the actin molecule, were found to be the most susceptible to oxidation to the sulfoxide derivative. Met-176, Met-190, Met-227, and Met-269 are the other sites of the oxidative modification. The increase in fluorescence associated with the binding of 8-anilino-1-naphtalene sulfonic acid to hydrophobic regions of the protein reveals that actin surface hydrophobicity increases with oxidation, indicating changes in protein conformation. Structural alterations were confirmed by the decreased susceptibility to proteolysis and by urea denaturation curves. Oxidation of some critical methionines (those at positions 176, 190, and 269) causes a complete inhibition of actin polymerization and severely affects the stability of actin filaments, which rapidly depolymerize. The present results would indicate that the oxidation of some critical methionines disrupts specific noncovalent interactions that normally stabilize the structure of actin filaments. We suggest that the process involving formation of actin carbonyl derivatives would occur at an extent of oxidative insult higher than that causing the oxidation of some critical methionine residues. Therefore, methionine oxidation could be a damaging event preceding the appearance of carbonyl groups on actin and a major cause for the functional impairment of the carbonylated protein recently observed both in vivo and in vitro.

Human intestinal cell monolayers are preferentially sensitive to disruption of barrier function from basolateral exposure to cholic acid: correlation with membrane transport and transepithelial secretion.

Unconjugated bile acids such as cholic acid cause diarrhoea, mucosal irritation and toxicity. We sought to define the mechanism of cholate permeation across intestinal mucosal cells to understand how cellular exposure and accumulation are deleterious to mucosal function. Human intestinal Caco-2 and T84 cell monolayers were prepared by high-density seeding and cultured for >14 days on permeable culture supports. Cholate transport and cellular accumulation were determined using [3H]cholic acid. Epithelial barrier function was assessed by measuring transepithelial electrical resistance (Rt) and [14C]mannitol fluxes. Exposure of Caco-2 epithelia to serosal cholate caused a dose- and time-dependent disruption of barrier function. Apical exposure was without disruptive effect. Similar responses were observed for T84 epithelia. Cholate was preferentially accumulated across the basolateral surfaces in both Caco-2 and T84 cells, but was subject to active transepithelial secretion in Caco-2 monolayers only. Net secretion was substantially reduced by ATP depletion, showed saturation kinetics, and was subject to competitive inhibition by other bile acids. Cholate secretion was also sensitive to inhibition by the leukotriene antagonist MK-571 but not by digoxin, suggesting that MRP2, not MDR1, was responsible. RT-PCR and Western blotting confirmed MRP2 expression in Caco-2 epithelia but indicated its apparent absence from T84 cells.

Increased cGMP levels mediate oxidant-induced disruption of microtubule (MT) cytoskeleton and increased permeability of monolayers of human intestinal cells

Increased cGMP levels mediate oxidant-induced disruption of microtubule (MT) cytoskeleton and increased permeability of monolayers of human intestinal cells

Increased cGMP levels mediate oxidant-induced disruption of microtubule (MT) cytoskeleton and increased permeability of monolayers of human intestinal cells

Increased cGMP levels mediate oxidant-induced disruption of microtubule (MT) cytoskeleton and increased permeability of monolayers of human intestinal cells

Hormonal regulation of oligopeptide transporter pept-1 in a human intestinal cell line.

The intestinal oligopeptide transporter (cloned as Pept-1) has major roles in protein nutrition and drug therapy. A key unstudied question is whether expression of Pept-1 is hormonally regulated. In this experiment, we investigated whether insulin has such a role. We used a human intestinal cell monolayer (Caco-2) as the in vitro model of human small intestine and glycylglutamine (Gly-Gln) as the model substrate for Pept-1. Results showed that addition of insulin at a physiological concentration (5 nM) to incubation medium greatly stimulates Gly-Gln uptake by Caco-2 cells. This stimulation was blocked when genistein, an inhibitor of tyrosine kinase, was added to incubation medium. Studies of the mechanism of insulin stimulation showed the following. 1) Stimulation occurred promptly (30-60 min) after exposure to insulin. 2) There was no significant change in the Michaelis-Menten constant of Gly-Gln transport, but there was a nearly twofold increase in its maximal velocity. 3) Insulin effect persisted even when Golgi apparatus, which is involved in trafficking of newly synthesized Pept-1, was dismantled. 4) However, there was complete elimination of insulin effect by disruption of microtubules involved in trafficking of preformed Pept-1. 5) Finally, with insulin treatment, there was no change in Pept-1 gene expression, but the amount of Pept-1 protein in the apical membrane was increased. In conclusion, the results show that insulin, when it binds to its receptor, stimulates Gly-Gln uptake by Caco-2 cells by increasing the membrane population of Pept-1. The mechanism appears to be increased translocation of this transporter from a preformed cytoplasmic pool.

Adhesion of Helicobacter pylori to polarized T84 human intestinal cell monolayers is pH dependent

Epithelial cells, which form tight polarized monolayers on porous substrates, constitute ideal model systems to study bacterial adhesion and invasion. The binding of Helicobacter pylori to the apical membrane of T84 cells, an epithelial cell line derived from a human colon carcinoma, was assessed biochemically and morphologically. Attachment was rapid, and binding remained constant over time, with a significant (P < 0.01, Mann-Whitney U test) ca. fourfold increase at pH 5.4 (76% +/- 22%) compared with pH 7.4 (18% +/- 7%). In contrast, adhesion of enteropathogenic Escherichia coli was not enhanced at pH 5.4. The transepithelial electrical resistance of the T84 cell monolayers was not affected by pH or by H. pylori. Following binding, H. pylori induced a reorganization of the brush border as reflected by actin condensation, facilitating the intimate association of the bacteria with the apical plasma membrane. H.pylori was not internalized, as shown by confocal microscopy. Some bacteria, found in deep invaginations of the apical membrane, were probably inaccessible to gentamicin, thus accounting for the observed tolerance to the antibiotic. These data provide the first evidence that an acidic environment favors Helicobacter adhesion and that binding is followed by survival of the survival of the bacteria in pockets of the apical membrane.

Adhesion of Helicobacter pylori to polarized T84 human intestinal cell monolayers induces a reorganization of the brush border and is PH dependent

Adhesion of Helicobacter pylori to polarized T84 human intestinal cell monolayers induces a reorganization of the brush border and is PH dependent

Acid‐base transport systems in a polarized human intestinal cell monolayer: Caco‐2

Acid-base transport systems have been incompletely characterized in intact intestinal epithelial cells. We therefore studied the human cell line Caco-2, cultured on Teflon membranes to form confluent monolayers with apical microvilli on transmission electron microscopy and progressive enrichment in microvillar hydrolases. Monolayers (16- to 25-day-old), loaded with the pH-sensitive dye BCECF-AM (2',7'-bis (carboxyethyl)-5-carboxyfluorescein), were mounted in a spectrofluorometer cuvette to allow selective superfusion of apical and basolateral surfaces with Hepes- or HCO(3-)-buffered media. Intracellular pH (pHi) was measured by dual-excitation spectrofluorimetry; calibration was with standards containing nigericin and 110 mM K+ corresponding to measured intracellular [K+] in Caco-2 cell monolayers. In HCO(3-)-free (Hepes-buffered) media, bilateral superfusion with 1 mM amiloride or with Na(+)-free media reversibly inhibited pHi recovery from an intracellular acid load (NH4Cl pulse) by 86 and 98% respectively. Selective readdition of Na+ to the apical or basolateral superfusate also induced a pHi recovery, which was inhibited by ipsilateral but not by contralateral amiloride (1 mM). The pHi recovery induced by apical Na+ readdition had a Michaelis constant (Km) for Na+ of 30 mM and a relatively high inhibitor constant (Ki) for amiloride of 45.5 microM. Initial pHi in HCO(3-)-buffered media was lower than in the absence of HCO3- (7.35 vs. 7.80). pHi recovery from an acid load in HCO3- was Na- dependent but was inhibited only 18% by 1 mM amiloride. The amiloride-independent pHi recovery was inhibited 49% by pre-incubation of cells in 5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). These data suggest that Caco-2 cells possess: (a) both apical and basolateral membrane Na(+)-H+ exchange mechanisms, the apical exchanger being relatively resistant to amiloride, similar to apical Na(+)-H+ exchangers in several normal epithelia; and (b) a Na(-)-dependent HCO3- transport system, either Na(+)-HCO3- cotransport or Na(-)-dependent Cl(-)-HCO3- exchange.