The Effect of Hyperosmosis on Paracellular Permeabilityin Caco-2 Cell Monolayers

Hitoshi INOKUCHI,1;y Takuto TAKEI,1 Katsuyoshi AIKAWA,1 and Makoto SHIMIZU2

1Oral Liquid Formulation R&D Laboratories, Self Medication Business, Taisho Pharmaceutical Co., Ltd.,1-403 Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan2Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received August 4, 2008; Accepted October 6, 2008; Online Publication, February 7, 2009

[doi:10.1271/bbb.80538]

The intestinal epithelium is a significant barrier tooral absorption of hydrophilic compounds, and theirpassage through the intercellular space is restricted bythe tight junctions. In this study we found that hyper-osmosis is a significant factor altering paracellulartransport in Caco-2 cell monolayers. Osmotic regula-tors, such as sodium chloride, mannitol, and raffinose,decreased transepithelial electrical resistance andenhanced lucifer yellow permeability. The effect ofthese osmotic regulators on Caco-2 cell monolayers wasnot likely to be caused by gross cytotoxicity. Althoughcertain amino acids and oligosaccharides have beenreported to have specific tight junction-modulatingactivity, we found that the increased paracellularpermeability of Caco-2 monolayers induced by thesecompounds was at least partly due to the increasedosmotic pressure of the test solutions. These findingsprovide a new potential precaution in the evaluation ofparacellular permeability-modulating substances usingthe Caco-2 cell monolayer system.

Key words: paracellular permeability; hyperosmosis;tight junction; Caco-2 cell

The intestinal epithelium represents a major barrier tothe absorption of orally administered nutrients and drugsinto systemic circulation. Translocation of nutrientmolecules across the intestinal epithelium occurs bypassive diffusion via the transcellular or the paracellularroute, or through carrier-mediated active or facilitatedtransport. The intercellular space of the intestinalepithelium restricts the passage of molecules due tothe presence of junctional complexes (tight junctions,intermediate junctions, and desmosomes).1,2) Hence theintestinal epithelium is a significant barrier to hydro-philic molecules because they cannot easily traverse thelipid bilayer of the cell membrane, and their passagethrough the intercellular space is restricted by tightjunctions, viz., the zonula occludens.

The paracellular transport route is an important routeacross the intestinal epithelial cell layers for hydrophiliccompounds, which are not transported by any carrier-mediated mechanism. The effects of anisotonic solutions

(mainly hypotonic solution) on Caco-2 and HT-29.cl19A intestinal epithelial cells have been reportedto enhance the transport of hydrophilic model com-pounds across the monolayers.3) In the rat jejunum,passive transfer of solutes like 2-deoxyglucose andmannitol, which is mostly done through the paracellularpathway, is modified by osmolality.4) Moreover, it ispossible that a paracellular route plays a central role inwater movements across this epithelial barrier in theCaco-2 cells after exposure to serosal hypertonicity.5)

On the other hand, osmotic stress is a proinflammatorysignal in Caco-2 cells, suggesting that an osmosenserexists in intestinal epithelial cells.6) This suggests thathyperosmolality affects the function of Caco-2 cells. Toour knowledge however, there have been only fewreports on the effects of hyperosmolality on paracellularpermeability in Caco-2 cell monolayers.Regulation of paracellular permeability also occurs in

response to Naþ-coupled transport of glucose and aminoacids across the apical membrane of intestinal cells,and is accompanied by alterations in cytoskeleton.7,8)

Previous studies have found that in response to theessential amino acid L-tryptophan at luminal concen-trations, which is to be superphysiological, theseresponses are so exaggerated as to induce disruption oftight junctions and trans-epithelial macromolecularleaks.9) Although several factors participate in theregulation of intestinal permeability, the mechanismsof control of intestinal permeation have not yet beenfully established.10)

Caco-2 cell lines derived from human colon adeno-carcinoma are capable of enterocytic differentiation andare regarded as a valid model of the human smallintestinal epithelium system.11–13) The fully differenti-ated and polarized Caco-2 cell monolayers are charac-terized by typical brush border membranes and tightjunctions.14) Caco-2 cell monolayers grown on perme-able membrane filters are used to determine the para-cellular permeability of hydrophilic compounds.The purpose of this study was to examine the effect of

the osmotic regulators (hyperosmosis) sodium chloride,mannitol, and raffinose on paracellular permeability incultured human intestinal cell monolayers. Furthermore,

y To whom correspondence should be addressed. Fax: +81-48-663-1045; E-mail: hitoshi.inokuchi@po.rd.taisho.co.jpAbbreviations: DFAIII, difructose anhydride III; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HBSS, Hank’s balanced

salt solution; LDH, lactate dehydrogenase; LY, lucifer yellow; NEAA, non-essential amino acids; PBS, phosphate buffered saline; PCC, palmitoylcarnitine chloride; TEER, transepithelial electrical resistance

Biosci. Biotechnol. Biochem., 73 (2), 328–334, 2009

we investigated the effects of several food factors,including ones already reported to have specific tightjunction-modulating activity, on the paracellular perme-ability of Caco-2 cell monolayers, together with the roleof osmotic pressure. Since the mucosal surface of theproximal small intestine, which is lined with a low-resistance epithelium, experiences transient mucosalosmotic loads during the process of normal digestion,it is important in human nutrition to determine the effectof hyperosmosis on drug absorption in human-derivedCaco-2 cell monolayers.

Materials and Methods

Materials. The Caco-2 cell line was purchased from the American

Type Culture Collection (Rockville, MD). Dulbecco’s modified

Eagle’s medium (DMEM), Hank’s balanced salt solution (HBSS),

palmitoyl carnitine chloride (PCC), and FITC-phalloidin were from

Sigma (St Louis, MO). L-glutamine, fetal bovine serum (FBS), non-

essential amino acids (NEAA), trypsin-EDTA, and penicillin–strepto-

mycin (10,000 units/ml and 10mg/ml in 0.9% sodium chloride) were

from Gibco (Gaithersburg, MD). Lucifer yellow (LY), Triton X-100,

sodium chloride, mannitol, raffinose, paraformaldehyde, L-glutamate,

L-serine, difructose anhydride III (DFAIII), and a LDH-Cytotoxic Test

kit were from Wako Pure Chemical Industries (Tokyo). L-Arginine,

L-glutamine, glycine, L-lysine, L-methionine, L-proline, and L-trypto-

phan were from Ajinomoto Healthy Supply (Tokyo). L-Valine was

from Kyowa Hakko Kogyo (Tokyo). All other chemicals were of

reagent grade.

Cell culture. Caco-2 cells were cultured in 57-cm2 plastic dishes

with a culture medium consisting of DMEM, 10% FBS, 1% NEAA,

2mM L-glutamine, 100 units/ml penicillin, and 100mg/ml streptomy-

cin. The cells were incubated at 37 C in a humidified atmosphere of

5% CO2 in air, and the culture medium was renewed on alternate days.

When they reached confluence, the cells were passaged at a split ratio

of 4 to 8 by trypsinization with trypsin-EDTA. All the cells used in this

study were between passages 55 and 60. A transport experiment was

performed using Caco-2 cells that had been cultured at a density of

1:6 105 cells/well in a 12-well collagen-coated Transwell insert

(12mm in diameter and 0.4mm pore size, Corning, Corning, NY). The

cell monolayers for the transport experiments were used after 21 d of

culture.

Transepithelial electrical resistance measurement. The integrity of

the Caco-2 cell monolayers was evaluated by measuring the trans-

epithelial electrical resistance (TEER) value. Measurements of TEER

across Caco-2 cell monolayers were performed using, a Millicell ERS

instrument (Millipore, Bedford, MA). Resistance due to the cell

monolayers was determined in the presence and the absence of the

compounds after subtracting the contribution of the blank filter and the

HBSS. The osmolality of the test solutions was measured by Advance

Osmometer Model 3D3 (Advance Instruments, Norwood, MA).

Lucifer yellow transport. Transport studies were performed on

filter-grown Caco-2 cell monolayers with LY (100mg/ml). An

experiment without a sample added to the apical side was performed

as a control. Fresh HBSS was applied to the both sides of the

Transwell. After incubation for 30min, HBSS containing the sample

(0.5ml) was added to the apical side of the cells, and HBSS (1.5ml)

was added to the basolateral side. After a 90min incubation period,

transport rates were determined by measuring the fluorescence

associated with the lucifer yellow present on the basolateral side.

The LY in the basolateral solution was determined fluorometrically at

430 nm for excitation and 540 nm for emission (SpectraMax Gemini

XS; Molecular Devices, Sunnyvale, CA).

Lactate dehydrogenase assay. To evaluate the damage to the Caco-

2 cells, lactate dehydrogenase (LDH) assay was performed following

Konishi et al.15) The Caco-2 monolayer, which had been cultured in

24-well plates for 21 d, was washed with 500ml of the HBSS and then

incubated with 500 ml of the HBSS containing the sample to be tested

at 37 C for 90min. The sample solution (supernatant) was removed,

and then 500ml of 0.1% Triton X-100 was added to each well to

solubilize the residual cells. The amounts of LDH in the supernatant

and the solubilized cell fraction were determined by the enzymatic

method with an LDH-Cytotoxic test kit. Cytotoxicity to the Caco-2

monolayers was expressed as the relative amount given by the ratio of

LDH released into the supernatant to the total amount of LDH, and this

latter amount was given by the sum of intracellular LDH and LDH

released by the control cells.

Fluorescence staining of the cell monolayers. The actin filament

structure in the Caco-2 cell monolayers was stained as previously

described.16) Cover glasses with cell monolayers were gently rinsed

with phosphate buffered saline (PBS), and then fixed with 4%

paraformaldehyde for 30min. The glasses were then washed 3 times

with PBS, treated with 0.1% Triton X-100 for 5min to increase

membrane permeability, washed 5 times with PBS, and then dried. The

dried glasses were treated with 1 mM FITC-phalloidin for 1 h and then

washed 5 times with PBS. Fluorescence staining was visualized with a

TCS-SP5 confocal laser scanning microscope (Leica Microsystems,

Wetzlar).

Statistical analysis. Experimental values are given as means and

S.D. Statistical comparisons were made by Dunnett’s multiple

comparison. A P < 0:05 value was considered to have statistical

significance.

Results and Discussion

Effects of osmotic regulators on TEER and LYpermeability across Caco-2 monolayersThe paracellular transport route is an important route

across intestinal epithelial cell layers for hydrophiliccompounds that are not transported by any carrier-mediated mechanism. This pathway is restricted by thetight junctions on the apical side of the epithelialcells.17,18) Previous studies indicate that the permeabilityof the paracellular route is modified by osmolality. Toour knowledge, however, there have been only fewreports on the effect of hyperosmolality on paracellularpermeability in Caco-2 cell monolayers. Since sodiumchloride, mannitol, and raffinose have been used asosmotic regulators,3,6,19) we investigated the changes inTEER and LY transport across Caco-2 monolayers afteradding these osmotic regulators to the apical chamber.Clearly, a dose-dependent decrease in the TEER value

was observed upon application of the osmotic regulators(Fig. 1A). The amount of LY that permeated from theapical to basolateral side of the cell monolayers alsoincreased (Fig. 1B). Although sodium chloride was mosteffective in these osmotic regulators, there was apositive linear correlation between LY and osmolality,whereas a negative linear relationship was observedbetween TEER and osmolality in Caco-2 cell mono-layers (Fig. 2A and B). Hence it is possible thatparacellular transport in the cell monolayers increasedosmolality-dependently. These results are consistentwith the findings that hypertonic solution (600mOsm)decreases TEER of Caco-2 cell monolayers of humanorigin.3) Exposure of cell layers to a hyperosmoticsolution immediately results in cell shrinkage to bringthe cytosol in osmotic equilibrium with the surroundingsolution.20) This contractile state might contribute to anincrease in the paracellular permeability of Caco-2 cellmonolayers.

Paracellular Permeability and Hyperosmosis in Caco-2 Cells 329

The cytotoxic effects of osmotic regulators on Caco-2cell monolayers

Our results indicate that osmotic modulators cause adecrease in TEER and an increase in permeability toLY, as has been observed for other agents that act viamodulation of tight junctions, such as sodium caprateand dodecylphosphocholine.21,22) Disruption of Caco-2cells by osmotic regulators might however account forthe increased permeability of the cell monolayers.Therefore, the cytotoxic effect of sodium chloride,mannitol, and raffinose on Caco-2 cells was examinedby LDH assay.

The various osmotic regulators exhibited little cyto-toxicity (LDH release) (Fig. 3). This indicates that thehyperosmosis-mediated increase in the permeability wasnot due to cell damage but to enhancement of theparacellular permeability of the cell monolayers. Todetermine whether the effect of osmotic pressure onCaco-2 cell monolayers is reversible, apical HBSScontaining osmotic regulators was replaced with freshHBSS after 90min of treatment. TEER returned to nearthe control value upon removal of sodium chloride(0–100mM) as measured after 18 h (Fig. 4A). Similarly,the TEER values with mannitol and raffinose recovered

to near the control values (Fig. 4B and C). The recoveryrate of TEER was dependent on the concentration of theregulators (osmolality), and the TEER values of themonolayers treated with 200mM osmotic regulators did

0

0.01

0.02

0.03

0.04

0.05

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Concentration (mM)

LY p

erm

eabi

lity

(nm

ol/m

in/c

m2 )

**

**

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0 50 100 200

Concentration (mM)

TE

ER

(%

)

**

**

**

**

**

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**

A

B

Fig. 1. Effects of Osmotic Regulators on TEER Value and LYPermeability.Caco-2 cell monolayers was incubated for 90min at 37 C in the

presence of up to 100mM sodium chloride, mannitol, and raffinose,and TEER value (A) and LY permeability (B) were measured.Values are mean S.D., n ¼ 3. Significantly different from 0mM

(p < 0:01, Dunnett’s test). , sodium chloride; , mannitol;, raffinose.

R2 = 0.810

0

0.01

0.02

0.03

0.04

0.05

200 300 400 500 600 700

Osmolality (mOsm/kg)

LY p

erm

eabi

lity

(nm

ol/m

in/c

m2 )

R2 = 0.900

0

20

40

60

80

100

200 300 400 500 600 700

Osmolality (mOsm/kg)

TE

ER

(%

)

A

B

Fig. 2. Relationships between Osmolality and Permeability Parame-ters.

Correlation for osmolality and TEER (A), and osmolality and LYpermeability (B) after 90min at 37 C incubation with osmoticregulators are shown. Values are means, n ¼ 3. , sodium chloride;, mannitol; , raffinose.

0

20

40

60

80

100

0 50 100 200

Concentration (mM)

LD

H r

elea

se (

%)

Fig. 3. Cytotoxic Effects of Osmotic Regulators on Caco-2 Cells.The amount of LDH released into the supernatant with respect to

the total amount of LDH was measured after incubating the Caco-2cells at 37 C for 90min with sodium chloride, mannitol, andraffinose. Data values are expressed as amounts relative to control,and are presented as the mean S.D., n ¼ 3. , sodium chloride;, mannitol; , raffinose.

330 H. INOKUCHI et al.

not recover to the control values in 18 h of incubationwith fresh HBSS. In order to confirm the effect ofhyperosmosis on the cells, F-actin, constituting thecytoskeleton, was stained with FITC-phalloidin. Noclear difference was seen between the photographs of thecytoskeletal structure incubated with the isotonic solu-tion (Fig. 5A) and cells incubated apically with 200mM

of sodium chloride, mannitol, and raffinose (Fig. 5B, Cand D). These results suggest that the actin filamentswere not affected by hyperosmosis. Actin filamentsattach directly to the cytoplasmic surface of the tight

junction, and perturbation of them causes paracellularbarrier disruption.1,23) These results indicate that thepromotive effects of osmotic regulators on paracellularpermeability are controlled, and do not result indisruption of the tight junctions on the monolayers.Hyperosmotic stress induces inflammatory cytokineproduction in human epithelial cells.24,25) Exposure ofrat cerebral endothelial cells to cytokines induced adecline in the TEER.26) Therefore, the increased para-cellular permeability and duration of low TEER causedby high osmotic pressure can be at least partly due to thecytokine production by Caco-2 cells. Moreover, it ispossible that passive paracellular absorption across thesmall intestine is modified by changes in luminal contentcomposition (osmotic pressure).

Effects of palmitoyl carnitine chloride, difructoseanhydride III, and amino acids on the TEER value inCaco-2 cell monolayersSeveral agents, such as PCC and DFAIII, have been

found to enhance paracellular transport.27,28) Previousstudies by Madara et al.7,8) showed that Naþ-coupledtransport of amino acids across the apical membrane ofintestinal absorptive cells increases the paracellulartransport of hydrophilic compounds by modulating thetight junctions. Since our present study suggests thatparacellular transport in Caco-2 cell monolayers isosmolality-dependent, the effects of osmolality onenhancement of paracellular permeability by knownenhancers (PCC and DFAIII) and amino acids wereinvestigated. Eight amino acids were selected withrespect to their polarity.The changes in TEER after application of the samples

and the osmolality of the sample buffer solutions areshown in Table 1. A dramatic decrease in the TEERvalue was observed on treatment of the Caco-2 cell

0

100

200

300

400

500

600

0 5 10 15 20

Incubation time (h)

TE

ER

(Ω

cm2 )

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100

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700

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900

0 5 10 15 20

Incubation time (h)

TE

ER

(Ω

cm2 )

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500

600

700

0 5 10 15 20

Incubation time (h)

TE

ER

(Ω

cm2 )

B

A

C

Fig. 4. Reversibility of TEER in Caco-2 Cell Monolayers afterTreatment with Sodium Chloride.The Caco-2 cells were treated with various concentrations of

sodium chloride (A), mannitol (B), and raffinose (C) for 90min at37 C, then the sample buffer solution was replaced with fresh HBSSand the TEER value was measured over time. Values are means,n ¼ 3. , 0mM; , 50mM; , 100mM; , 200mM.

A

C

B

D

Fig. 5. Effects of Osmotic Regulators on the Actin-Filament Struc-ture in Caco-2 Cell Monolayers.

Caco-2 cell monolayers cultured with 0mM osmotic regulator (A),200mM sodium chloride (B), 200mM mannitol (C), and 200mM

raffinose (D) on cover glasses were stained with FITC-phalloidin for1 h and analyzed with a confocal laser scanning microscope.Bar = 25 mm.

Paracellular Permeability and Hyperosmosis in Caco-2 Cells 331

monolayers with PCC. The osmolality of the samplebuffer with PCC was almost same as that of the controlbuffer, indicating that this TEER decrease was not dueto a change in osmolality. Hochman et al.27) reportedthat transport enhancement due to PCC was accompa-nied by an increase in apical membrane permeabilityand a reduction in cell viability. The sample solutionswith DFAIII and L-glutamate also significantly de-creased the TEER value of the Caco-2 cell monolayer(Table 1). However, in contrast to PCC, DFAIII andL-glutamate solutions showed comparatively highosmolality due to the high concentration (100mM) ofDFAIII, and increased the salt concentration of theL-glutamate solution after neutralization with NaOH.The TEER-decreasing effect of DFAIII was strongerthan that of L-glutamate, although the osmotic pressuresof DFAIII and L-glutamate were almost the same. Thismight indicate that the lower TEER value induced by theDFAIII solution can be attributed not only to theosmolality change but also to other mechanisms. Suzukiand Hara28) reported that DFAIII increased net calciumtransport in cells, and that a rise in [Ca2þ]i was involvedin the opening of tight junctions. In eight amino acids, adecrease in the TEER value was observed only when theCaco-2 cell monolayers were treated with L-glutamate.The TEER-decreasing activity of amino acids is likely tobe independent of the polarity of amino acids. Ourfindings indicate that L-tryptophan did not decrease theTEER value of Caco-2 cells. In a previous study,L-tryptophan elicited a decrease in transepithelial resist-ance in the hamster small intestinal epithelium.9) Thisdifference might have been due to the colonic originof Caco-2 cells: some transport systems are expressedto a lesser extent in Caco-2 cells than in normalenterocytes.29)

Effects of L-glutamate on the permeability of theparacellular route in Caco-2 cell monolayers

Our findings indicate that L-glutamate decreased theTEER value of the Caco-2 cell monolayers. It has beenreported that the excitatory amino acid transporter 1transcript was consistently expressed in the Caco-2 cellline and that L-glutamate transport was clearly Naþ-dependent.30) Although, it has been suggested that the

presence of actively Naþ-cotransported substratesincreases the permeability of the paracellular route,7,8)

the exact mechanism by which the epithelial barrierfunction is decreased by L-glutamate remains unclear.The decrease in TEER in the Caco-2 cell monolayersdue to the 50mM L-glutamate solution was accompaniedby the comparatively high osmotic values of theL-glutamate solution. We investigated the effect ofosmotic pressure on enhancement of paracellular per-meability by L-glutamate.TEER decreased dose-dependently with the applica-

tion of L-glutamate. LY permeability also increased ina dose-dependent manner (Fig. 6A and B). However,the osmotic values of the test solutions containingL-glutamate (0mM, 320mOsm; 50mM, 411mOsm; 75mM, 457mOsm; 100mM, 505mOsm) were thought to behigh enough to increase the permeability of the para-cellular route, as shown in Fig. 2. The osmotic pressureof L-glutamate rose when NaOH was used as the pHcontrol. The effect of L-glutamate on the paracellularpermeability of the Caco-2 cell monolayers underisotonic conditions is shown in Fig. 7. In a low osmoticHBSS prepared with a reduced NaCl concentration,L-glutamate (0mM, 173mOsm; 50mM, 265mOsm;75mM, 310mOsm; 100mM, 352mOsm) did not affectthe TEER value of the monolayers. Therefore theincreasing effect of paracellular permeability due to

Table 1. Effects of PCC, DFAIII, and Amino Acids on TEERValuesin Caco-2 Cell Monolayers, and Osmolality of Test Solutions

Concentration

(mM)

Osmolality

(mOsm/kg)

Relative

TEER (%)

Control 0 291 98 4

PCC 1 302 8 4

DFAIII 100 381 57 10

L-Tryptophan 50 339 103 3

L-Glutamate 50 382 80 6

Glycine 50 334 109 5

L-Glutamine 50 344 106 3

L-Methionine 50 337 101 8

L-Proline 50 340 99 2

L-Serine 50 338 102 7

L-Valine 50 344 100 7

PCC, DFAIII , and amino acids were added to the buffer solutions and

osmolality was measured. The Caco-2 cells were then incubated with each

test solution for 90min at 37 C and the TEER value was measured. Values

are mean S.D., n ¼ 4. Significantly different from the control (p < 0.01,

Dunnett’s test).

0

0.01

0.02

0.03

0 20 40 60 80 100

LY p

erm

eabi

lity

(nm

ol/m

in/c

m2 )

**

**

0

20

40

60

80

100

120

0 20 40 60 80 100

L-Glutamate (mM)

TE

ER

(%

) **

**

**

A

B

L-Glutamate (mM)

Fig. 6. Effects of L-Glutamate on TEER Values and LY Permeability.Caco-2 cells were incubated with L-glutamate for 90min at 37 C,

and TEER (A) value and LY permeability (B) were measured.Values are mean S.D., n ¼ 3. Significantly different from 0mM

(p < 0:01, Dunnett’s test).

332 H. INOKUCHI et al.

L-glutamate observed in this study was probablydependent on the osmotic pressure instead of Naþ-coupled transport. Since the osmotic pressures ofbreak>L-glutamate and DFAIII were almost the samein this study, hyperosmosis might partially contribute tothe enhancement of paracellular permeability byDFAIII. In the paracellular transport assay using Caco-2 cell monolayers, not only the effect of the testsubstance but also the effect of osmotic pressure mightbe evaluated. Therefore, in the evaluation of paracellularpermeability-modulating substances, it is necessary totake into account the osmotic pressure of the testsolutions.

In conclusion, the present study provides evidencethat hyperosmosis enhances paracellular permeabilitywithout damaging cell membranes. Although villusosmolality during glucose exposure has been reportedto be in a range of 600–1,000mOsm,31–34) further studiesare needed to verify the occurrence of a similar responseto apical hyperosmosis in the native intestinal epithe-lium in vivo and to clarify the intracellular mechanism.Further studies are needed to verify the occurrence of asimilar response to apical hyperosmosis in the nativeintestinal epithelium in vivo and to clarify the intra-cellular mechanism. We suggest that not only the testsubstance but also the osmotic pressure of the testsolutions affects the paracellular permeability of Caco-2cell monolayers. Hence it is necessary to pay attentionto the osmotic pressure of test solutions when highconcentrations of test samples are used. Our studyprovides a new potential precaution in the evaluation ofparacellular permeability-modulating substances in theCaco-2 cell monolayer system.

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0

20

40

60

80

100

120

0 25 50 75 100

L-Glutamate (mM)

TE

ER

(%

)

**

**

**

Fig. 7. Effect of Hyperosmosis on L-Glutamate-Mediated Decreasein TEER in Caco-2 Cell Monolayers.Caco-2 cells were incubated with HBSS ( 125mM, 50mM

sodium chloride) containing L-glutamate for 90min at 37 C, and theTEER value was measured. Values are mean S.D., n ¼ 3.Significantly different from 0mM (p < 0:01, Dunnett’s test).

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